Index

A

Abbe, Ernst 181, 187, 485, 493absolute molecule mass 4absolute quantification (AQUA) of

modified peptides 660absorptionbands, ofmost biologicalmolecules 139measurement 140–142of photon 135spectroscopy 131

acetic acid 228acetonitrile 227N-acetyl-α-D-glucosamine

(αGlcNAc) 579acetylated proteinsdetection of 651separation and enrichment 649

acetylation 224, 645–647sites, in proteins 656identification of 655

N-acetyl-β-D-glucosamine(βGlcNAc) 579

N-acetylgalactosamine 129achromatic objectives 183acid-base properties 5, 234acidic and basic acrylamide

derivatives 261acidic native electrophoresis 257acidic peptides 562acidification 3activation domain (AD) fusion

proteins 385activation energy 37, 38acylation 109, 110adeno-associated viruses 913adenosine triphosphate (ATP) bindingpocket 1050site 1048

Aequorea victoria 182, 192aerosols, danger of contamination 770affinity capillary electrophoresis

(ACE) 285–286binding constant, determined by 285changing mobility 286complexation of monovalent

protein–ligand complexes 285affinity chromatography 91, 268, 650affinity purification mass spectrometry

(AP-MS) 381, 1003agarose concentrationsDNA fragments, coarse separation

of 692migration distance and fragment

length 692agarose gels, advantages of 260agglutination 72aggregation number 288AK2-antibodies 102alanine 562alanine-scanning method 870albumin 3, 995alcohol dehydrogenase (ADH) gene

promoter 382alkaline phosphatase (AP) 87, 741, 746,

753alkylation 32of cysteine residues 210

allele-specific hybridization probes 774allergies 65allotype determinations 80all-trans conformation 48amidation 110amino acid analysis 118, 207, 301, 313biophysical properties of 887identification of 316–317liquid chromatography with optical

detection systems 303

post-columnderivatization 303–305

pre-columnderivatization 305–308

reagents used for 310sample preparation 302acidic hydrolysis 302alkaline hydrolysis 303enzymatic hydrolysis 303

using mass spectrometry 309L-α-amino acid residues/termini 225amino acid sequence analysismilestones in 319problems 322amino acids 323–324background 324initial yield 324modified amino acids 324purity of chemicals 324sample to be sequenced 322–323sensitivity of HPLC system 325

state of the art 3256-aminoquinoyl-N-hydroxysuccinimidyl

carbamate (ACQ) 307ammonium sulfate 10Amoeba dubia 785AMPD anion 747amyotrophic lateral sclerosis (ALS)

1059analogue sensitive kinase alleles (ASKAs)

approach 1047, 1048kinases 1050

analogue-sensitive kinases, inhibitors andcofactors 1049

analytical method, developmentof 234–235

analysis of fractionations 238column efficiency, optimization

of 235

Bioanalytics: Analytical Methods and Concepts in Biochemistry and Molecular Biology, First Edition.Edited by Friedrich Lottspeich and Joachim Engels. 2018 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2018 by Wiley-VCH Verlag GmbH & Co. KGaA.

analytical method, developmentof (continued )

fractionation 237–238retention factors, optimization

of 235–236scaling up to preparative

chromatography 236–237selectivity, optimization of 235

analytical ultracentrifugation 409–410basics of centrifugation 411–412photoelectric scanner in 410principles of instrumentation 410sedimentation velocity

experiments 412experimental procedures 413–414N-acetyl-L-glutamate-kinase and signal

protein PII, interactionbetween 414–415

physical principles 412–413sedimentation–diffusion equilibrium

experiments 415–416anilinothiazolinone (ATZ) amino

acid 316antibiotic resistance genes 914antibodies 63, 64, 1034allotype 65binding 87in conjunction with use of natural

Fcγ-effector functions 104engineering 99–102, 101Fc-receptor 65handling of 68–69microarrays 956production 98properties of 64–65IgA 64IgD 65IgG 64, 65IgM 64, 65

as reagents 64types of 98–99

antibody-directed cellular cytotoxicity(ADCC) 104

antigen–antibody-complexesin vitro, reversible separation of 65

antigen–antibody reaction 64, 71–72antigenic system 74antigen interaction at combining

site 67–68antigens 69–71antisense oligonucleotides 959, 961,

963, 964antisense probein vitro synthesis of 899

ApA-wedge/B-junction models 832aperture diaphragm 183apochromatic objectives 183apolipoprotein B 3apoptosis 645

aptamers 869, 971high-affinity RNA/DNA-

oligonucleotides 971–974selection of 971–973Selex procedure 869uses of 973–974

aqueous DNA solutionsphenolic extraction of 666

Ardenne, Manfred von 486arginine-rich motif (ARM) 859aromatic amino acid 225, 561Arrhenius’ equation 44aryl azides 125aryl(trifluoromethyl)diazirines 126asparagine 579Aspergillus orizae 862, 863, 896, 899atomic force microscope (AFM) 486,

519detection of ligand binding and

function of 526determining protein complex assembly

and function by 524functional states and interactions of

individual proteins 526–527gap junctions 523imaging with 521interaction between tip and

sample 521–522mapping biological

macromolecules 522–524preparation procedures 522schematic representation of 520single molecules 524–526

atomic orbitals (AOs) 134attenuated total reflection (ATR) 166automated projection spectroscopy

(APSY) 463autoradiogram 791autoradiography 652avian myeloblastosis virus (AMV) 761avidin–biotin complex formation (ABC

system) 87azido salicylic acid 125

B

Bacillus amyloliquefaciens 683Bacon, Roger 181bacterial artificial chromosomes

(BACs) 788, 934bacterial suspension 672Bacto tryptone 671band broadening 222, 223barcode array 955base-catalyzed β-elimination 651base pairing, complementarity of 720Baumeister, Wolfgang 486B-cell 99, 100Beckman Optima 410

benzophenonederivatives 123photolabels 127

benzoyl cyanide (BzCN) 865p-benzoyl-L-phenylalanine 123bicinchoninic acid assay 27–28bifunctional reagents 121, 122binding tests 73, 85, 86, 99binocular tubes 184biochemical pathwayscomplex structures, representation

of 1024oxygen demand 425–426

bioinformatics analysis 219biological functions, alteration of 97–98biologically relevant lipids, classification

of 614biological starting materials, disruption

methods 9bioluminescence resonance energy

transfer (BRET) 408BioMed Central Bioinformatics 877biomimetic recognition elements 419biomimetic sensors 427aptamers 428molecularly imprinted

polymers 427–428biophysical methods 131biopolymers 131biosensors 419, 428anti-interference principle 424concept of 420–421construction/function 421–423coupled enzyme reactions in 424enzymatic analyte cycles 424enzyme electrodes

generation 423–424sequence/competition 424

BIO systemdetection system 746labeled nucleotides 742

biotinbiotinylation, reagents for 128biotinyl groups 129disadvantage of 746labeled dNTPs structure 743

biphasic column reactor, sequencerswith 321–322

bis(1,10-ortho-phenanthroline)-copper (I)complex (OP-Cu) 849

bispecific antibodies 100bis(trimethylsilyl)trifluoroacetamide

(BSTFA) 621bisulfite methylation analysis 819, 821bisulfite PCRenzymes for restriction analysis 822RASSF1A-promoter 822restriction analysis 820–822, 821

bisulfite-treated DNA 820

1086 Index

amplification and sequencingof 819–820

biuret assay 26BLOSUM 62, 887mutation data matrix 888

blotting 22. see also electroblotting;nucleic acid blotting

capillary blots 706dot blotting unit 707membranes 705

blue native polyacrylamide gelelectrophoresis 259

Bolton–Hunter reagent 109bonding energy 37Borries, Bodo von 486“bottom up” protein analysis 314bovine serum albumin (BSA) 24, 54Bradford assay 28branched DNA (bDNA) 752amplification method 782

Bravais lattices 535brilliant blue 251Broglie, Louis de 4865-bromo-4-chloro-indoxyl phosphate (BCIP)NBT, coupled optical redox

reaction 7485-bromo-UTP (BrU)structures of 906

bruton tyrosine kinase (BTK) 1049bufadienolide K-strophanthin 745buffer systems 255substance 43–44

bump-and-hole method 1047t-butyldimethylsilyl ether

(TBDMS) 710

C

Caenorhabditis elegans 942, 967, 1015,1052

caesium chloride (CsCl)density gradient 703, 721solutions 14

caged compounds 111Ca2+ imaging 205calcium phosphate 913calibration curve 24, 25calorimetry 47Candida albicans 768cap analysis of gene expression

(CAGE) 868capillary blots 706capillary columns 621capillary electrochromatography

(CEC) 288–289capillary electrophoresis (CE) 244, 275,

281–295, 296, 299, 649, 650, 827basic principles 277detection methods 279–281

fluorescence detection 280mass spectrometrydetection 280–281

UV-detection 280engine, electroosmotic flow 278–279historical overview 275–276Joule heating 279sample injection 277electrokinetic injection 277hydrodynamic injection 277stacking 277

schematic view 276setup 276–277capillary 276–277instrumental setup 276voltage unit 276

capillary gel electrophoresis(CGE) 290–291, 701, 711

sifting media for 290capillary isoelectric focusing

(CIEF) 291–294focusing withchemical mobilization 292–293pressure/voltage mobilization 292

one-step focusing 292capillary zone electrophoresis

(CZE) 281–285, 561, 1027buffer additives 284–285capillary coating 284electrodispersion 282ionic strength 283optimization of separation 283peak broadening 282pH-value of buffer 283temperature 283

carbene forming reagents 126carbodiimides 125carbohydrates 5725-carboxylcytosine (5caC) 817carboxymethylaspartic acid 233carboxypeptidases 328cleaved amino acids, detection 328polypeptides, degradation 327specificity of 327

carotenoids 638carrier ampholyte IEFadvantages/disadvantages 261properties for ideal carrier

ampholyte 261catalysts 37cationic detergent electrophoresis 258cDNA libraries 385cell adhesion 64cell arrangements 1039cell disruption 7cell isolation 96cell sensors 425cellular immunology 95–97cellulose esters 16

centimorgans (cM) 926, 929centrifugation 9, 11, 411basic principles 12density gradient 14CsCl solutions 14sucrose 14

fractionation of separated bands 14rotors for 11techniques 12differential 12–13isopycnic 14zonal 13–14

ceramides 641cetyl(trimethyl)ammonium bromide

(CTAB) 258, 279, 670chain termination method 789, 790channel electron multiplier (CEM) 356chaotropic reagents 7charged coupled device (CCD)cameras 144, 497, 544, 1059sensor 808

chemical biology 1041innovative chemical approaches to

study biologicalphenomena 1041

multidisciplinary approach 1042chemical crosslinking, reagents

properties 866chemical diversity 224chemical genetics 1046protein–protein interaction stabilizer

fusicoccin 1046small organic molecules, for protein

function modulation 1042ASKA technology 1050–1051bump-and-holeapproach 1047–1050

cyclic process 1042forward and reverse 1046–1047study of 1044–1046switching biologicalsystems 1051–1052

chemical labeling reactions 734chemical nucleases, structure of 849chemical protein 107chemical reactions, rate of 36–37chemical shifts 459chemiluminescence 652detection, of hydroperoxy lipids

621substrates 745

chimeric antibodies 100chiral MEKC 289principle 290

chiral separations 289chloramphenicol acetyltransferase

(CAT) 911, 914N-chlorobenzenesulfonamide 33chlorobutane 316

Index 1087

4-chloro-7-nitrobenz-2-oxa-1,3-diazole 118

chlorophyll–protein complexes 152cholesterol, lipophilic molecules 969chromatin epigenetic modifications

analysis 828chromatin immunoprecipitation (ChIP)assay 826, 850, 895chip analysis (ChIP chip) 939ChIP-on-chip analysis 952

chromatin-immuno-precipitationsequencing (ChipSeq) 813, 828

chromatin modifications 828chromatograms 220, 221chromatographically incompletely

separated componentsESI spectra 716

chromatographic dimension, peak-picking module 1008

chromatographic material, binding to 17chromatographic separation modes 220,

224employed in peptide and protein

separation and 233high-performance affinity

chromatography (HP-AC) 233high-performance aqueous normal

phase chromatography(HP-ANPC) 230

high-performance hydrophilicinteraction chromatography(HP-HILIC) 229–230

high-performance hydrophobicinteraction chromatography(HP-HIC) 230–232

high-performance ion exchangechromatography(HP-IEX) 232–233

high-performance normal-phasechromatography(HP-NPC) 228–229

high-performance reversed-phasechromatography(HP-RPC) 227–228

high-performance size exclusionchromatography 227

for peptides and proteins 225–226chromatographic traces 1007chromophores in biological

macromoleculeselectronic absorption properties

of 154chromoproteins 148chromosomal in situ suppression

(CISS) 919chromosomebreakage sites, physical markers 927conformation capture techniquechromosomal interactions 829

distance between genes 925interaction analyses 828–829

chymotrypsin 24, 207circular dichroism (CD) 178–180cyclic peptides 56715mer peptides with typical α-helical

conformation 566spectroscopy 564

circular polarized light 133Clark, L.C. 429clone-based mapping procedures 936cloned gene, riboprobe creation 900cloning systems 934, 935positional cloning 938reference system 937

cloud 875CLUSTAL alignment 892clustered regulatory interspaced short

palindromic repeats(CRISPR) 974

CMC. see critical micellar concentration(CMC)

CNBr cleavage 108coefficients of variation (CVs) 1003coherent light 184co-immunoprecipitation

(co-IP) 398–399cold-shock proteins (CSPs) 860ColE1 multi-copy plasmids, of

enterobacteriaceae family 671collision energy (CE) 1005collision induced dissociation

(CID) 357–358, 999column efficiency 222, 234combinatorial molecular phenotypes

(CMPs)motif 1063with proteins 1061

combined bisulfite restriction analysis(CoBRA) 820

comparative genomic hybridization(CGH) 747, 917, 921–924, 951

chromosomal 922exemplary demonstration 923hybridization and data

acquisition 923intermixture 921–922microarray 920, 922–924normalization 921probes 918

competitive inhibitors 40–41competitive (RT) PCR 765, 766competitive radioimmunoassay

(RIA) 82, 83complementarity determining regions

(CDRs) 101complementary DNA (cDNA) 761, 843complementary target sequences 758complement fixation 94–95

complete MOTIFs 885complex 3D data sets, analysis of 514combination of EM and x-ray

data 514–515flexible fitting 515hybrid approach 514identifying protein complexes in

cellular tomograms 515–516rigid body docking 515segmenting tomograms and

visualization 515complex protein mixtures, quantification

of 24computer-aided analysis 413concentration 17condenser 184conditional protein splicing 1054confocal high-speed-spinning disk

systems (Nipkow systems) 199confocal laser scanning microscopy

(cLSM), principle 198confocal spinning disk microscopy

(Nipkow) 199principle 199

trans conformation 48contour-clamped homogenous electric

field (CHEF) method 698, 699contrast transfer function

(CTF) 499–501, 501–503cooling curves 54cooling rates 49Coomassie Brilliant Blue G250 28Coomassie staining 252spot analysis 985

Coons, Albert 182copper ion 151Cotton effect 564coumarins 119coupling liquid chromatography (LC)and mass spectrometry (MS),

advantage 375cover slip 184CpG adjuvants 709CpG dimer 948CpG island 688CpG-methylation 817critical micellar concentration

(CMC) 18, 59, 288crosslinked protein 129crosslinking factor 250, 251crossover-electrophoresis 78cryo-electron microscopy 496, 497cryo-electrontomography(CET) 486,517cryopreservation 69crystallization 3crystallographic R-factor 543crystallographic unit celland crystal packing 534

crystallography 529

1088 Index

crystals, and x-ray diffraction 533–538C-terminal sequence analysis 325chemical degradation methods 325degradation of polypeptides with

carboxypeptidases 327detection of the cleaved aminoacids 328

specificity ofcarboxypeptidases 327

peptide quantities and quality ofchemical degradation 327

Schlack–Kumpfdegradation 325–327

cw-EPR spectroscopy 468–469cyan fluorescent protein 406cyanoethyl adducts, formation 711cycle sequencing 795cyclic peptides 224cyclodextrins 289cyclotron-movement 349Cy-dyes 986cysteines 656acetylated 655residues, chemical modification of 210

cystic fibrosis (CF) 767cytochromes 149–151cytogenetic methods 917–924cytolytic T-cellseffector cells, activation of 105recruitment of 105

cytomegalovirus (CMV) 772DNA virus 965

cytoplasmic RNA 677migration of 698

cytosine methylation 817cytosine, to uracil 819cytotoxicity 64

D

dabsyl chloride (DABS-Cl) 116, 307dansyl derivatives, optical properties

of 117dark field microscopy 188data dependent acquisition (DDA) 1000peptide quantification 1002

data independent acquisition(DIA) 1000, 1008

data interpretation 369data mining 1030–1031DAVID database 878Dcm-methylation 817deamidation 224decipher regulatory cis-elements 907van Deemter-Knox equation 222delta restrictions cloning 787denaturationdenaturing high-performance liquid

chromatography (dHPLC) 940

DNA sequencing, gel-supportedmethods 792

proteins 7in situ hybridization 919

Denny–Jaffe reagent 126deoxynucleotide triphosphates 759depletion 982depurination reaction 710detection methods 189chemical staining 189direct and indirect immunofluorescence

labeling 190–191fluorescence labeling 189–190for live cell imaging 191

fluorescence microscopy, fluorophores/light sources 193–194

histological staining 189incandescent lights 194labeling with quantum dots 192lasers 195mercury vapor lamps (HBO

lamps) 195mercury–xenon vapor lamps 195physical chemistry of staining

(electro-adsorption) 189physical staining 189types of light sources 194in vitro labelingwith fluorescent fusion proteins(GFP and variants) 192–193

with organic fluorophores 191, 192xenon vapor lamps 195

detergents 19chromatographic support materials for

separation of 21–22ionic 20micelles, formation of 18nonionic 20properties of 18and removal 18removal of 20–22zwitterionic 20

diafiltration 16dialysis 15, 17diamagnetic biomolecules 466diastereomers 710diatomic molecule, vibration properties

of 163diazopyruvoyl compounds 126dideoxy method 7892´,3´-di-deoxynucleotide 789dielectric coefficient 247diethyl pyrocarbonate (DEPC) 864chemical formula/mechanism 676

difference gel electrophoresis(DIGE) 252, 270–271,986

internal standard 271minimal labeling 270–271

principle 271saturation labeling 271

different extraction methods 21differential centrifugation 12–13differential equation 36differential interference contrast 186differential RNA-seq (dRNA-seq) 868differential scanning calorimeters

(DSCs) 47, 48, 54curve 50–53design 49instrument requirements 48–49

diffraction phenomena and imaging 187Digitalis lanata 745Digitalis purpurea 745digoxigenindetection system 745digoxigenin:anti-digoxigenin (DIG)hapten 753system 744

structure of 7444-(N,N-dihexadecyl) amino-7-nitrobenz-

2-oxa-1,3-diazole (NBD-dihexadecylamine) 619

dihydrorhodamine (DHR) 962,5-dihydroxybenzoic acid (DHB) 1066N,N´-diisopropylcarbodiimide

(DIC) 558dilution 154,5-dimethoxy-2-nitrobenzyl

chloroformate 111dimethylformamide (DMF) 558dimethyl sulfate (DMS) 846modification 849

dimyristoyl-phosphatidylglycerol 60dinitrophenol (DNP) 7492,4-dinitrophenol (DNP) 742

diode array photometer 142dioxetane chemiluminescence

reaction 748diphenylthiourea (DPTU) 315, 316diphosphatase 793dipolar coupling 473direct agglutination 72direct blotting electrophoresis

(DBE) 741disc electrophoresis 255–257discontinuous polyacrylamide gel

electrophoresis (discPAGE) 244

principle of 256discovery proteomics 996disruption method 9disulfide bonds , cleavage of 209–210dithioerythritol (DTE) 685dithiothreitol (DTT) 126, 209, 269, 685DNA analysis 957adenine methyltransferase 851associated protein modifications 828

Index 1089

DNA analysis (continued )backbone 801binding motifs 835binding proteins 828, 951calcium phosphate crystals 913copying enzyme 846double helix 720footprint analysis 844scheme of 849

fragments 721, 833helix parameters 832hybridization 161, 948hydrolysis method 827methylation analysis 827–828

length standards 693with methylation specific restriction

enzymes 823–825molecule 689non-viral introduction 912oligonucleotides 730, 732, 736, 843,

896, 953, 962DNA chip technologies 429, 1035DNA complexity, cot value 721DNA Data Bank of Japan (DDBJ) 875DNA:DNA hybrids 722DNA–DNA interactions 1036DNA library 675DNA methylation 818, 895, 949, 950with methyl-binding proteins 826specific restriction enzymes 824

DNA methyltransferases (DNMTs) 817DNAmicroarray technologies 939, 945,

953, 1036barcode identification 954–955beyond nucleic acids 956–957DNA analysescomparative genomic hybridization(CGH) 951

genotyping 948methylation studies 948–949protein–DNAinteractions 951–952

sequencing 949–951DNA synthesis 952on-chip proteinexpression 953–954

RNA synthesis 953RNA analyses 946splicing 947structure/functionality 947–948transcriptome analysis 946–947

structural analyses 956universal microarray

platform 955–956DNA-modifications, analysis of 818DNA polymerase 207, 429, 798advantage of 756

DNA polymorphisms 927DNA–protein binding partner 870

DNA-protein complex 837, 839, 867chemical reagentsdiethyl pyrocarbonate (DEPC)847

dimethyl sulfate (DMS) 846halo-acetaldehydes 847hydroxyl radicalfootprint 847–848

KMnO4/OsO4 846–847N-ethyl-N-nitrosourea (ENU) 847

gel analysis of 838interactions 857DNA curvature 832–833DNA topology 833–834double-helical structures 831–832

DNA-RNA hybrid 961DNase-I digest 845, 918DNA sequencing 785automated capillary 799device 799energy transfer dyes 797fluorescent markings 796gel-free methods 806adaptations to library preparation/sequencing 810

classic pyrosequencing 807illumina sequencing 809–810indexed libraries 811mate-pair library creation 810–811multiplex DNA sequencing 805paired-end-sequencing 810pyrosequencing, principle of 807454-technology (Roche)807–808

microdrop PCRgenomic sequencing/resequencing 812–813

semiconductorsequencing 811–812

sequencing technologies,applications 812

tag counting–RNASeq/ChipSeq 813

target (exome) enrichment 811next generation sequencing

(NGS) 786principle of 799

single molecule sequencing 813nanopore sequencing 814native hemolysin pore, inmembrane 814

PacBio RS single molecule 814single molecule real time(SMRT) 814

DNA sequencing, gel-supportedmethods 786

autoradiogram 791chain termination method, to

Sanger 790

chemical cleavage, according toMaxam/Gilbert

cleavage reactions 801–804end labeling 800–801A G-cleavage reaction 802Maxam–Gilbert method 800multiplex DNAsequencing 804–805

RNA sequencing 805–806solid phase process 804α-thionucleotide analogues804

thymine- and cytosine-specificfission reaction 803

cycle sequencingprinciple of 795with thermostable DNApolymerases 795–796

denaturation/neutralization 7922´-deoxynucleotide 790deoxynucleotide analogues structure, in

dideoxy sequencingreactions 794

dideoxy method 7892´,3´-di-deoxynucleotide 789diphosphatase 793final denaturation 795labeling techniques/verification

methodsautomated samplepreparation 798–800

energy transfer dyes 797fluorescent labeling/onlinedetection 797

internal labeling, with labeleddeoxynucleotides 798

isotopic labeling 796–797labeled terminators 798online detection systems798

primer labeling 797–798primer hybridization 792–793strand synthesisand additives 794cofactors 794nucleotide analogs 794–795pyrophosphorolysis 793

T7 DNA polymerase 791–792,796

dot blotting unit 707dot immunoassay 88double beam spectrometer 143double-stranded RNA-binding domain

(dsRBD) 860Down’s syndrome 719doxorubicin 104doxycycline-inducible DNA-binding

unit 1051Drosophila embryos 720, 729

1090 Index

Drosophila melanogaster 785, 942,947, 967, 1016, 1052

dry chemistry 420DryLab G/plus 236dual-beam photometer 142Duchenne muscular dystrophy 767dyeenergy transfer 797laser-induced emission 797

dynodes 356

E

Eddy diffusion 222Edman degradation 116, 210, 313, 315,

564, 654amino acids, identification

of 316–317cleavage reaction of 316length of amino acid sequence 319phosphorylated and acetylated amino

acids, localization of 654quality of 317–319reactions of 315–316repetitive yield 317–319

Edman, Pehr 313Edman sequencing 108EDTA-containing buffer 845Egger, David 182Einstein’s formula 412Ekins, Roger 1034elastic scattering 171electric field vector 134electric ion traps 348–349electroblotting 272blot membranes 273blot systems 272semidry blotting 272–273tank blotting 272

transfer buffers 273electrochemical luminescence

markers 742electrodiffusion 78electroelution from gels 263–264electroendosmosis 247, 248electrokinetic injections 277–278electromagnetic radiation 132, 133electromagnetic waves 132, 134electron crystallography 486electronic DNA biochips 428–429electronic transition 133electron microscope, imaging

process 183, 485, 492, 498analytical electron

microscopy 494–495electron beam with object, interactions

of 493–495electron energy lossspectroscopy 494

imaging and information contentof 493–494

mass determination 494Fourier transformation 499–501perspectives of 516–517with phase plate 495–496pixel size 498–499resolution 492–493scanning electron

microscopy 494–495electron nuclear double resonance

(ENDOR) 477–478experiments 477–478

electron paramagnetic resonance(EPR) 119

applications for 479local pH values 480mobility 480–481quantification of spin sites andbinding constants 479–480

basics 467comparison EPR/NMR 481–482cw-EPR spectroscopy 468electron spin and resonance

condition 467–468electron spin nuclear spin

coupling 469–470g-value 469hyperfine anisotropy 470–472significance 481spectroscopy of biological

systems 466electron spin echo envelope modulation

(ESEEM) 475–476problems 475three-pulse spectrum 476

electron spin–electron spincoupling 472–473

anisotropic component 473electron spin nuclear spin coupling

(hyperfine coupling) 469–470cw-X-band EPR spectrum of TPA in

liquid solution 470Fermi contact interaction 469

electron tomography of individualobjects 512–514

electron transfer dissociation (ETD) 999fragmentation sources 992

electropherogram 296electrophoresis 243, 690–691capillary gel electrophoresis

(CGE) 701–702DNA fingerprinting 696DNA, gel electrophoresis ofagarose gels 691circular DNA, separation of 692denaturing agarose gels 693–694double-stranded DNAfragments 691–692

double-stranded DNA, non-denaturing PAGE of 694

low melting agarose/sievingagarose 694

native polyacrylamide gels,abnormal migrationalbehavior 694–695

oligonucleotidepurification 696–697

polyacrylamide gels 694practical considerations 692–693protein–DNA complexes, non-denaturing gels 694

single-stranded DNA(SSCP) 695–696

gel media for 250pulsed-field gel electrophoresis

(PFGE) 698–700applications 699–700principle 698–699

RNA, gel electrophoresis 697formaldehyde gels 697RNA standards 697–698

two-dimensional gel electrophoresis(2D gel electrophoresis) 700

of DNA 700–701of RNA 700temperature gradient gelelectrophoresis (TGGE) 701

electrophoretic mobilities 253electrophoretic mobility shift analysis

(EMSA) 836electrophoretic separation

techniques 243historical review 244media 243separation principle 243theoretical fundamentals 245–248

electrospray ionization (ESI) 335, 998,1064

charged residue model 337interfaces, types of 281ion emission model (IEM) 337ionization, principle of 335–338macroscopic/microscopic 336mass spectra, properties of 339–341mass spectrometry 220, 234MS/MS instruments 377MS spectrum, of metabolically

labeled 1015sample preparation 341schematic structure and variants of

electrospray sources 339source and interface 338–339spectra 715chromatographically incompletelyseparated components716

components detection 716

Index 1091

electrospray ionization mass spectrometry(ESI-MS) 228, 711

electrospray mass spectrometryhydrophobic transmembrane peptide

fragment 565purified 16mer peptide amide 565

electrostatic attraction 60electrostatic interactions 60ellipticity 179Ellman’s reagent 112EMBL Nucleotide Sequence

Database 786emulsion-PCR reaction (emPCR) 807ENCODE project 882, 883, 939endoplasmic reticulum 129, 579endoprotease AspN 985endoprotease LysC 985endoproteinases 328endothermic processes 48endothermic transition 48, 49energy levels, transitions between 137energy transfer group systems 797enthalpy 35, 49, 59, 147Entrez database system 878entropy 35, 36, 147enzymatic activity 44enzymatic digestion 313enzymatic DNA synthesis 952enzymatic labeling 735of DNA 736nick translation 736PCR amplification 736random priming 736reactions 735reverse transcription 736of RNA 736–737terminal transferase reaction 736

enzymatic methods 651as catalysts 37–38

enzyme-catalyzed DNApolymerization 794

enzyme-controlled reactions, rate of 38enzyme diaphorase (DP) 753enzyme immunoassays (EIA) 91–93Biacore technique 93–94

enzyme-linked immunosorbent assay(ELISA) 745, 1033

signal amplification 751enzyme–substrate complex 39enzyme–substrate interactions 1038enzyme sulfurylase catalyzes 807epidermal growth factor 119epidermal growth factor receptor

(EGFR) 1041epifluorescence microscope 188epigenetic modifications, analysis

of 817DNA replication 817

epitope mappingz 70

Epstein-Barr virus (EBV) 785, 913Er-YAG-laser 331erythropoietin (EPO) 581amino acid sequence with 588GluC peptides, theoretical/

experimental masses of 589, 590RP-HPLC/ESI-MS, total ion

current 588schematic representation 582

Escherichia coli 385, 683, 785, 933,934, 953, 978

cell 856cytosine 825DNApolymerase holoenzyme 736DNApolymerase I 736promoter sequences 883

ethidium bromide 253chemical structure of 702geometric properties of circular

DNA 703ethyl acetate 316ethylcyanoglyoxylate-2-oxime

(Oxyma) 558ethyl trifluorothioacetate 110eukaryotic cellscytoplasmic RNA 697

eukaryotic DNA 721eukaryotic mRNA species 678European Bioinformatics Institute

(EBI) 875European Molecular Biology laboratory

Open Software Suite (EMBOSS)analysis package 881PEPSTATS routine 882

sequence analysis, on web 881–882E-value 890evaporative light scattering detectors

(ELSD) 620exon sequencing 940exonuclease III (Exo III) 845exothermic heat 54expressed protein ligation (EPL) 1052,

1053expressed sequence-tagged (EST) 776extracted ion chromatogram (EIC) 713,

717

F

Fab fragment 745Faraday cup 357schematic design and operating

principle 357far-Western blot 399–400FASTA format 879, 880Fayyad, U. 1029Fc-engineering 101Fenton reactionhydroxyl radicals 847

Fe 1-(p-bromoacetamidobenzyl)-ethylenediaminetetraacetic acid(Fe-BABE)

structure of 850Ferguson plot 253field inversion gel electrophoresis

(FIGE) 698filamentous phages 676finger-printing methods 787Fischer, Emil 573five-stranded β-sheet 460flame ionization detectors (FID) 620florescence resonance energy transfer

(FRET) 851flow cell 266flow velocities 2229-fluorenylmethoxycarbonyl (FMOC)

chloride 306–307fluorescamine 304fluorescein 119, 797fluorescein:anti-fluorescein (FLUGS)

system 742, 746fluorescein cadaverine 119fluorescein isothiocyanate (FITC) 116,

1059fluorescein-tagged

phosphoramidites 851fluorescence 652action spectroscopy 157coded beads 1034detection methods 653detector 220DNA chips 429energy transfer 117labeling 116, 119with dansyl chloride 118with fluorescamine 118with phthaldialdehyde inthiols 118

methods 651microscope 486 (see also

fluorescence microscopicanalysis, special)

principles of 188fluorescence correlation spectroscopy

(FCS) 160, 202–203, 856, 1042measurement 856

fluorescence energy resonance transfer(FRET) 749

fluorescence in situ hybridization(FISH) 729

fluorescence labeling, with fluoresceinisothiocyanate (FITC) 118

fluorescence lifetime imaging microscopy(FLIM) 160, 204–205, 406

fluorescence loss in photobleaching(FLIP) 202

fluorescence microscopic analysis,special 186, 188, 197–198, 1064

1092 Index

confocal high-speed-spinning disksystems (Nipkow systems) 199

light microscopic super resolutionbelow Abbe limit 200–201

live cell imaging 199–200measurement of movement of

molecules 202Ca2+ imaging 205fluorescence correlationspectroscopy (FCS) 202–203

fluorescence lifetime imaging(FLIM) 204–205

fluorescence loss in photobleaching(FLIP) 202

fluorescence recovery afterphotobleaching (FRAP) 202

fluorescent-speckle microscopy(FSM) 203

Förster resonance energy transfer(FRET) 203–204

Raster image correlationspectroscopy (RICS) 203

spectral unmixing 204multiphoton fluorescence

microscopy 198–199NSOM/SNOM (near-field scanning

optical microscopy) 202PALM (photoactivated localization

microscopy) 201(S)SIM ((saturated) structured

illumination microscopy) 201stimulated emission depletion

(STED) 201stochastic optical reconstruction

microscopy (STORM) 201total internal reflection fluorescence

microscopy (TIRFM) 202fluorescence quenching 119fluorescence recovery after

photobleaching (FRAP) 108,160, 202

fluorescence resonance energy transfer(FRET) 381, 402–403, 852–853

bioluminescence resonance energytransfer (BRET) 408

fluorescent probes for 406–407FRET based on fluorescence lifetime

measurements 406FRET estimation based on sensitized

acceptor emission 404–405FRET estimation using acceptor

photobleaching 405–406genetically encoded FRET pairs—

caveats and challenges 407–408instrumentation for intensity-based

FRET measurements 404key physical principles 403methods of measurements 403–406

fluorescence spectroscopy 154

emission and action spectra 156–157frequent mistakes in 161–163fluorescence ofcomplexes 162–163

FRET overdone 162GFP overdone 162incomplete/wrong labeling withfluorophores 161

quantum dots overdone 162shading and inner filter effect I 162shading and inner filter effect II 162

principles 154–156fluorescence staining 252fluorescence studies using intrinsic and

extrinsic probes 157–158fluorescence techniques 131fluorescent activated cell sorter (FACS)

apparatus 96fluorescent antibodies 116fluorescent detection 744fluorescent DNA hybridization 917absorption vs. emission-spectra 920DNA probes 918fluorescent hybridization signals,

evaluation of 920labeling strategy 917–918DNA probes 918–919nick translation 919PCR labeling 919random priming 919

signals, absorption vs. emission-spectra 920

in situ hybridization 919denaturation 919hybridization 919pre-annealing 919stringency 919

fluorescent in situ hybridization(FISH) 742, 917, 936, 940

of genomic DNA 920–921interphase-FISH/fiber-FISH 921metaphase 921multicolor 920–921

fluorescent labeling 9182´-deoxynucleotides

(fluorescein-15-dATP) 798terminators 798

fluorescent-speckle microscopy(FSM) 203

1-fluoro-2,4-dinitrobenzene 313fluorophores 119, 193, 194, 9504-methylumbelliferone (4-MU) 915

5-fluorouridine (FU)structures of 906in vivo labeling of nascent RNA 907

foam formation 617FokI 683advantage of 828

Folin–Ciocalteau phenol reagent 26

formic acid 560, 5615-formylcytosine (5fC) 817Förster distance 852Förster resonance energy transfer

(FRET) 108, 160–161,203–204

comparison between PELDORand 479

components 749efficiency (E) 406principle 728rate 161

Fourier analysis 500Fourier, Joseph 499Fourier transformation 350, 351,

499–501, 500, 501mass spectrometer 219

fragmentation techniques 296, 357collision induced dissociation

(CID) 357–358generation of free radicals (ECD,

HECD, ETD) 360–362photon-induced dissociation (PID,

IRMPD) 360prompt and metastable decay (ISD,

PSD) 358–360Franck–Condon principle 137, 154Fraunhofer scattering 142free amino acids, analysis of 303free electron laser (FEL) 529, 550free energy 35free flow electrophoresis 266–267principle 266

free working distance 183frozen-hydrated specimens, imaging

procedure for 496–497fundamentally different network

architectures 1030fusion proteins 182

G

galactocerebrosides 641Galilei, Galileo 181GAL4 system 869DNA-binding domain 868yeast Gal4 protein 381

g anisotropy 470–472gas chromatography (GC) 1027gas-phase fragmentation

approaches 992gas phase sequencer 321gauche-conformers 48gel chromatography 16gel electrophoresis 248, 806, 839, 982,

984gel retardation, background 836–838nucleic acids 841proteins separated 984

Index 1093

gel electrophoresis (continued )two-dimensionalprinciple and practicalapplication 700

gel filtration methods 21, 666, 667, 708gel-free sequencing methods 806gel phase 48gel retardation 837, 839, 840gel-supported DNA sequencing

methods 789GenBank flat-file formatted

database 880gene defects 719generation of free radicals (ECD, HECD,

ETD) 360–362genetic defects, detection of 773–776allele-specific PCR 775denaturing gradient gel electrophoresis

(DGGE) 776length variation mutations 773–774oligonucleotide-ligation assay (OLA)

technique 775restriction fragment length

polymorphisms 774reverse dot blot 774sequencing 774single-strand conformational

polymorphism (SSCP) 775–776genetic engineering 571genetic mapping 925disease genes 932genetic markers 927human genome 931–932, 942integration of 940–942linkage analysis 929χ2 test 929–930for plausibility 930–931

microsatellites 927physical mappingcloning systems 934, 935, 936genes, identification/isolationof 937–939

hereditary disease 940high-throughputsequencing 936–937

recombinant clones 934–935restriction of wholegenomes 932–934

STS mapping 935transcription maps, of humangenome 939–940

recombination 925–927restriction fragment length

polymorphisms (RFLPs) 927single nucleotide polymorphisms

(SNPs)/single nucleotide variants(SNVs) 927–929

genetic predisposition 932genome 610–611

genome-level regulatoryinformation 882

genome sequencing 610genome-wide association studies

(GWAS) 940genome-wide gene inhibition analysis,

schematic representation 955genomic loci 906genomic region, integrated map 941genotyping 948, 950to phenotype, organizational

structure 1025Gibbs free energy 241Gibbs–Helmholtz equation 61Giemsa staining 941G-less cassettes, for in vitro

transcription 909β-globin gene 765glucose disaccharides 572glucose enzyme electrodes 428–429D-glucose, stereochemistry of 573glutamate dehydrogenase 42glutathione S-transferase (GST)analyzing interactions in vitro,

GST-pulldown 397–398protein 398

N-glycanschemical shift 604composition analysis 600core structures 581coupling constants 605–606exoglycosidase digestion 602–6041H NMR spectroscopy 6041H NMR spectrum 607individual, analysis of 581–610isolation of individual

N-glycans 599–600linkage with peptide backbone 579mass spectrometry 604methylation analysis 600–602nuclear Overhauser effect

(NOE) 607–608pentasaccharide Man3-GlcNAc2

580pool, release and isolation

of 590–599spatial interaction of sugar

residues 609–610structural reporter groups 606structure 580types of 580

O-glycans structure 580–581glycerophospholipids (GP) 614glycine-glycine removal 994glyco-analytical studies 582glycolipids (GL) 614glycolysis 1024glycome 610–611goals 610

glycopeptides, mass spectrometricanalysis on basis of 588–590

glycoproteins 251, 258, 571, 572, 579,611

intact, analysis on basis of 582electrophoretic analyses 582–584monosaccharide components 585neutral monosaccharidecomponents 585–587

sialic acid 587using lectins 584–585

purification 572glycoproteome research, special challenge

of 610glycosidic bond 572, 574–578anomeric configuration 575, 576exo-anomeric effect 577–578linkage direction 577

glycosylation 224, 329, 571, 579O-glycosylation 579glycosyltransferases 579, 610glyoxal gels 697good laboratory practice (GLP)

conditions 611goodmanufacturing practice (GMP) 611G-protein coupled receptors 107guide RNA (gRNA) 974Gquery interface 878gradient elution 227, 236green fluorescent protein

(GFP) 158–159, 182, 406, 911,915, 1043

variants 193g-value 469

H

Hahn, Erwin 475Halobacterium salinarium 149, 523HapMap Project 932haptenantibodies 99dNTP 733structure of 743

H-bond donors 832heat capacity 49heating rates 49heavy metals 25height equivalent to one theoretical plate

(HETP) 222HEK293T cells 913helical B-DNA, structure of 832helical DNA conformations 832helix-loop-helix proteins (HLHs) 835helix-turn-helix structures (HTHs) 835hemagglutination 72, 73Henderson–Hasselbalch equation 44hepatitis B surface antigen

(HbsAg) 1035

1094 Index

hepatitis B virus (HBV) 772hepatitis C-virus (HCV) 752, 764, 772,

967heptafluorobutyric acid 228Herpes simplex 514heterogeneous amplification

systems 724heteronuclear NMR-spectroscopy 219hexaminidase 129higher-energy collisional dissociation

(HCD) 990, 999high field/high-frequency EPR

spectrometer 471–472high-performance anion exchange

chromatography (HP-AEX) 232high-performance aqueous normal phase

chromatography(HP-ANPC) 230

high performance cation exchangechromatography (HP-CEX) 232

high-performance gel permeationchromatography (HP-GPC) 227

high-performance hydrophilic interactionchromatography (HP-HILIC)229–230

high-performance hydrophobicinteraction chromatography(HP-HIC) 230–232

effect of salt cations 231protein–hydrophobic surface

interactions, enhanced by 232protein separations, selectivity of

230salts used in mobile phase 231

high-performance ion exchangechromatography (HP-IEX)232–233

high performance liquid chromatography(HPLC) 219, 220, 827, 1027

chromatogram of peptidemixture 564

column, operating ranges 237narrow bore 643separation of peptides by 561

high-performance normal-phasechromatography(HP-NPC) 228–229

high-performance reversed-phasechromatography(HP-RPC) 227–228

high-performance size exclusionchromatography (HP-SEC) 227

high-resolution two-dimensionalelectrophoresis 267–268

difference gel electrophoresis(DIGE) 270–271

internal standard 271minimal labeling 270–271saturation labeling 271

first dimension, IEF in IPGstrips 269–270

workflow 267prefractionation 268affinity chromatography 268fractionation according tocharge 269

subcellular components 268proteins, detection and identification

of 270sample preparation 268second dimension, SDS

polyacrylamide Gelelectrophoresis 270

high-throughput technology(hSHAPE) 866

high voltage transmission electronmicroscopes (HVTEMs) 493

HiSeq 810histologic stains 190histone acetyl transferases (HATs)enzymes 647internal lysine residues 646

histone deacetylases (HDACs) 646–647enzymes 647

histone H4 proteoforms 997Hoechst 33258 669Hofmeister series 10homing endonucleases genes

(HEGs) 683homogenization 7Hoogsteen base pairing 961Hooke, Robert 181, 485horizontal electrophoresis system 249house-keeping gene 764HPLC buffer 713HPLC chromatography 715HP-RPC purification 234hubs 1030Human Genome Project (HGP) 719,

785human immunodeficiency virus

(HIV) 772HIV-Rev protein 869provirus genomes 765trans-activator response element

(TAR) 869humanized antibody 100human proteinsmonoisotopic masses 989

human X chromosomephysical and genetic map 931

Huntington gene locus 773Huntington’s disease (HD) 773trinucleotide (CAG-) expansion 773

HybProbes 725, 728HybProbes 728hybrid antibodies 100hybrid instruments 351

hybridization methodsheterogeneous systems for qualitative

analysis 723heterogeneous systems for quantitative

analysis 723–724homogeneous systems for quantitative

analysis 725FRET system 728intercalation assay 728–729molecular beacon system 728in situ systems 729TaqMan/5´ nucleasemethods 725–728

hybridoma technique 99hydrogen bonds 169, 177hydrolysis methods, DNAmolecule 844DNA polymerases, exonuclease

activity of 845DNase I 844–845exonuclease III (Exo III) 845λ exonuclease 845

hydrolytic cleavage 313hydrophilic contaminants 15hydrophilic peptide 316hydrophilic proteins 20hydrophobic molecules 59hydrophobic peptides 5621-hydroxybenzotriazole (HOBt) 558hydroxyethyl cellulose (HEC) 701hydroxyl radical reactions 847, 8485-hydroxymethylcytosine (5hmC) 817hydroxypropylmethyl cellulose

(HPMC) 701N-hydroxysuccinimide (NHS) 125, 270N-hydroxysuccinimidyl-4-azidosalicylic

acid 125hypercholesterolemia 767hyperfine anisotropy 472hyperfine sublevel correlation experiment

(HYSCORE) 476–477pulse sequence 476splitting scheme 476theoretical HYSCORE spectrum 477

hypochromism 139hysteresis effects 54

I

identification, detection, and structureelucidation 368

identification 368–369structure elucidation 369–375verification 369

IgG antibody 828IgG immunoglobulinsantigen interaction at combining

site 67–68functional structure 66schematic structure and function 66

Index 1095

Illumina sequencing systems 809, 810exome enrichment 812

Ilyobacter tartaricus 523image analysis 498image cycler microscopy (ICM) 1058,

1060antibody based 1057

image isomerism, D-glucose/L-glucose 576

imaging cycler robots 1059–1063multi-pipetting unit, a fluorescence

microscope, and CCDcamera 1059

imidazole 33iminodiacetic acid 233immobilization 23, 88immobilized metal-chelate affinity

chromatography (IMAC)233, 649

on a membrane 17pH gradients 244reagents, analysis using 419

immune agglutination 73immune binding 84–88immune conjugates 104–105immune defense 63–64immune fixation 80immunoagglutination 72, 73application 72–73direct agglutination 72

immunochemical detection 651, 652immunocompromised AIDS

patients 965immunocytochemistry 90immunodetection 88immunodiffusion 75, 78one-dimensional simple

immunodiffusion ofOudin 75–76

two-dimensional immunodiffusion ofOuchterlony 77–80

immunoelectrophoresis 79immunofluorescence labeling 191, 196direct and indirect 190

immunoglobulins 995VL variable domain 892

immunohistochemistry 90immunopharmaceuticals, strategies for

production 103immunoprecipitating systems 73–75,

98, 649immunosensors 426–427immunotherapeutics 64industrial-scale sequence

production 875inelastic scattering 171informative markers 927infrared spectroscopy (IR) 163, 1026molecular vibrations 164–165

principles 163–164proteins, infrared spectra of 168–171technical aspects 165–168

inhibitors 40competitive 40–41non-competitive 41RNA (RNAi) molecules 953

in situ hybridization (ISH) 917in situ protein expression 954insoluble proteins 6instrumentation 319–322intact nuclei, isolation of 906Intelligent Systems in Molecular Biology

Conference (ISMB) 877interactomics–systematic protein–protein

interactionsDNA to protein

microarrays 1035–1036protein microarrays 1033–1034antibody–antigeninteraction 1037–1038

application of 1037–1039enzyme–substrate interaction 1038ligand–receptor interaction 1038reverse phasemicroarrays 1038–1039

sensitivity throughminiaturization–ambient analyteassay 1034–1035

interference 184contrast microscopy 188–189interfering substances 24

International HapMap Project 927interspersed repetitive sequences

(IRSs) 918hybridization with singular probes 918

intrinsically disordered proteins (IDP) 464in vivo proteolysis 207iodinations 33iodoacetamides 119, 125ion chromatograms 1008ion detectors 355ion exchange chromatography (IEX) 240ionic detergents 20ionic retardation 21ionic strength 43, 60ionization methods 329, 330ion mobility spectrometry (IMS) 378ion pairing reagent 236ion pair reversed-phase HPLC 712separation 714

ion-trap mass spectrometer 219isobaric tags for relative and absolute

quantitation (iTRAQ) 1020enzymatically cleaved proteome

states 1020isobaric reagents 1020

isocratic elution, optimization of 236isocyanate 316

isoelectric focusing (IEF) 244, 259–260carrier ampholytes 261cathodal drift 261electrode solutions 261immobilized pH gradients 261–262preparation of 262–263

measuring pH gradient 260pH gradients, kinds of 260principle 260separation media 260separator IEF 261titration curve analysis 263

isopycnic centrifugation 14isoschizomer EcoRII I-BstNI 825isoschizomers 824isotachophoresis (ITP) 293–295order of electrolytes and 294

isothermal titration calorimeters(ITCs) 48, 54, 58, 61

curve 55, 59, 60scheme 55

isothiocyanate, reaction with 111isotope-coded affinity tag 650isotope coded protein label

(ICPL) 1018–1019ICPLQuant 1019reagents 1018

isotopically labeled linker (ICAT) 660reagent 1016, 1017technique 1017

isotopic distributions 989isotopomer envelope 989isotopomers 987

J

Jablonski diagram 137–140JASPAR database 884, 885Joule heating 279

K

karyograms 917drawbacks of 917

Kepler, Johannes 181Ketenes 126K-homology domain (KH) 859RNA-binding motif 860

Kjeldahl method 25Klebsiella pneumoniae 768Klenow fragment 788, 791of DNA polymerase I 843

Knoll, Max 486Koch, Robert 182, 485Köhler, August 485Köhler illumination optimization 184Krebs cycle 1024Kyoto Encyclopedia of Genes and

Genomes (KEGG) 1024

1096 Index

L

labeled oligonucleotides 729label probesradioisotopes, characteristics of 739RNA probes, synthesis of 731

LabExpert 236laboratory information management

system (LIMS) 800lactate oxidase (LOD) score 931Lambert–Beer law 25, 140–142, 253Lamm’s differential equation 412, 415Larmor frequencies 474, 475laser, for fluorescence excitation 280laser microprobe mass analysis

(LAMMA) 1064N-Lauryl-sarcosine 678law of mass action 36, 416lectin-affinity chromatography 611Leeuwenhoek, Antoni van 181Leucine zipper proteins 835Leu2 gene 382ligase chain reaction (LCR) 751, 779–780LightCycler® 728, 756profiles 729

light emitting diodes (LEDs) 143, 144,157

light field diaphragm 184–185light field microscopy 187light induced co-clustering

(LINC) 408–409light microscopic technologies 183light scattering 144consequence for absorption

measurement 141linear combinations of atomic orbitals

(LCAO) 134linear dichroism (LD) 177IR spectroscopy 178

linear discriminant analysis(LDA) 1008

linear gradient gel, casting of 255linear polarized light 132linear polyacrylamide 701linear-solvent-strength theory 236Lineweaver–Burk plot 40, 41linkage analysis 929linoleic acid 621lipidation 224lipidome analysis 640–642, 641advantages of ESI-MS based 641biological sample, experimental

strategy for mass spectrometricanalysis 641

lipids 48, 613analysis of selected lipid classes 626fatty acids 627–628nonpolar neutral lipids 628–629polar ester lipids 630–633

whole lipid extracts 626–627biological functions 613combining analytical systems 623coupling of gas chromatography andmass spectrometry (GC/MS) 625–626

coupling of HPLC and massspectrometry (LC/MS) 625

coupling of HPLC and UV/Visspectroscopy 623–624

tandem mass spectrometry(MS/MS) 626

extraction 615hormones and intracellular signaling

molecules 633–638liquid phase extraction 616membranes 4methods for analysis 618chromatographicmethods 618–622

1H, 13C, and 31P NMRspectroscopy 623

immunoassays 622–623mass spectrometry 622UV/Vis spectroscopy 623

modified proteins,analysis 1052–1053

perspectives 642–643solid phase extraction 616–617structure and classification 613–615vitamins 638–640

lipofection 913lipoproteins 251liposomes 48, 50liquid chromatography (LC) 144, 220,

995, 998liquid chromatography tandem mass

spectrometry (LC-MS/MS) 999analysis 713bottom-up workflow 999high-throughput analysis 996instruments 376–377oligonucleotides 709mass spectrometricinvestigation 712–714

phosphorothioate oligonucleotide,IP-RP-HPLC-MS investigationof 714–717

principles of 709–711purity investigation/characterization 711–712

liquid handling system 798liquid-ordered state 48liquid phase sequenator 320Listeria monocytogenes 700live cell imaging 199–200liver alcohol dehydrogenase

(LADH) 174liver tissue

differential 1H ÍÌR spectroscopicanalysis 1027

living systems, hierarchical representationof complexity 1025

loading capacity 228locked nucleic acids (LNAs) 732probes 733

locus control regions (LCRs) 895Lowry assay 26–27LTYYTPEYETK 1015luciferase enzymes 748luciferin bioluminescence reaction 748luminescence 744Luria-Broth 671lymphocytes 65resistant to HIV 967

lysate 672LysC digest 1019lysine residues 108lysis 105

M

macromolecular crowding 515MAFFT 891magnetic bead isolation 679magnetic force microscope (MFM) 519forces between AFM tip and object 520principle 520

magnetic ion trap 349–355magnificationof lens 185total, of a microscope 186

major histocompatibility complex(MHC) 63

maleimides 119Malpighi, Marcello 181mammalian cells, in vivo analysis 571DNA transfer 912chemical-based transfection 913electroporation 913lipofection 913non-chemical methods 913transient/stable expression 913viral transduction 913

gene-regulatory cis-elements 911promoters 911–912

promoter activity 911marker chromosome 920mass analyzer 329, 341–343time-of-flight analyzers

(TOF) 343–345mass determination 362calculation of mass 362calibration 365derivation of mass 366determination of number of

charges 365–366influence of isotopy 362–365

Index 1097

mass determination (continued )problems 366–367signal processing and analysis 366

massively parallel sequencing(MPS) 785, 786, 806

mass spectrometry (MS) 108, 219, 220,315, 329, 394, 711, 982, 1064

accurate MALDI mass spectrometryimaging 1068–1069

achievable spatialresolution 1065–1067

amino acid analysis using 309aTRAQ-LC-MS-MS 309CE-MS 309direct infusion MS-MS 309GC-MS 309HILIC-MS 309ion pair-LC-MS-MS 309

analysis 397analytical microprobes 1064basic principles 1001coarse screening, by MS

imaging 1068components of 330glioblastoma tissue section, MALDI

images 1068identification/characterization

of 1069–1070lateral resolution/analytical limit of

detection 1067mass spectrometric pixel

images 1064–1065mouse spinal cord, SMALDI MS/MS

image of 1070mouse urinary bladder tissue section,

SMALDI images 1069peptide within, standard MALDI

preparation 1065phosphorylated/acetylated proteins,

detection of 653position informationmicroanalytical image 1065visual coding 1066

protozoa during mating, SIMS imagesof 1068

quantification 378–379sequencing of peptides, principle

of 371SIMS/ME-SIMS/cluster SIMS

imaging 1067SMALDI mass spectrometry imagingdried-droplet preparation 1067

techniques 866toponome mapping, by ICM 1064

mate-pair sequencing 811Mathieu equations 346, 347, 349matrix-assisted laser desorption ionization

(MALDI) 329, 1064, 1068instrument 345

mass spectra, characteristics of 332mass spectrometry (MALDI-MS)

330–331, 345, 643in biochemical analytics, typicalmatrix substances for 331

protein analysis 333sample preparation 332–335

matrix solution 296mouse spinal cord 1070peptide mass fingerprint (PMF) 1015proof-of-principle 1064sample preparation 334time of flight (MALDI-TOF)analysis 840mass spectrum, chemicallyacetylated histone H4 653

spectrometry 867spectrum, monoclonalantibody 333

spectrum, peptide angiotensinII 333

Maxam-Gilbert methodDNA sequence reaction 897reaction 846sequencing reaction 847

Maxam–Gilbert method 789, 800, 804,820

MaxQuant 1019Mbp1 protein 878mean proteins 24meiosis, recombination/crossing

over 926melting 47membrane-bound DNA probe 906membrane lipids 58membrane proteins 6, 82membrane–water interface 1192-mercaptoethanol 209, 685β-mercaptoethanol 126Merrifield, Bruce 555messenger RNA 128metabolic fingerprinting 1023metabolic labeling 660, 1015, 1016metabolic profiling 1023metabolite target analysis 1023metabolomics 1023, 1024analytic techniques, couplingwith separation technologies 1028

application 1032general strategies and

techniques 1027knowledge mining 1029–1030profiling 1027–1028and systems biology 1025–1026technological platforms 1026–1027

metabonomics 1023metalloproteins 151metarhodopsin I (MI) 149metarhodopsin II (MII) 149

methidiumpropyl-EDTA-Fe(MPE-Fe) 849

methionine 563methoxyamine hydrochloride 621methylated-CpG island recovery assay

(MIRA) 825methylated DNA immune-precipitation

(MeDIP) 826methylation analysiswith bisulfite method 819by DNA hydrolysis/nearest

neighbor-assays 827–828by methylcytosine-binding

proteins 825–826by methylcytosine-specific

antibodies 826–827principle of 820

methylation specific PCR(MSP) 822–823

principle of 822RASSF1A-promoter 823

methylation specific restriction enzymes,DNA analysis 823–825

methyl binding domain (MBD) 8255-methylcytosine 817methylcytosine-binding proteins,

methylation analysis 825–826methylcytosine-specific antibodies,

methylation analysis 826–827N,N´-methylenebisacrylamide 250MethyLight method 8234-methylumbelliferyl-β-D-

galactopyranoside(4-MUG) 915

micellar electrokinetic chromatography(MEKC) 286–288

capacity factor 286electrophoretic mobility 286principle 287time window 287

micelle building agents 288Michael addition 651Michaelis–Menten equation 39–41Michaelis–Menten theory 38–39microarray analyses 945microarray suitabilityDNA vs. proteins 1036

microbial sensors 425–426microcalorimetry 47microchannel plate 356–357microchip electrophoresis (MCE)

297miniaturization 298separation of amino acid derivatives by

chip-MEKC 298microelectromechanical systems

(MEMSs) 680micro RNAs (miRNAs) 856, 959,

970–971

1098 Index

microsatellites 927. see alsopolymorphic sequence taggedsites

microscopic stages 184microscopy techniques, biological

structures and suitable 485microspots sensitivity 1035micro-total analysis systems

(μ TAS) 297Mie scattering 142migration behavior, of superhelical 693MIRArecombinantGST-tagged MBD2b

protein 825miRNA-122, liver-specific 971mixed mode HILIC/cation-exchange

chromatography (HILIC/CEX) 229

MMLV RTase 761mobile phase 221, 222, 618salts used for HP-HIC 231

modern LC-ESI-MS systems 376modern microscopic techniques 485modulation transfer function (MTF) 497molar mass 4molar ratio 58molar transition enthalpy 51molecular orbitals (MOs) 134molecular probescaging gives temporal and spatial

control 1051molecular replacement (MR) 541–542molecular vibrations 164–165molecules binding, to membranes 58insertion and peripheral

binding 58–61molecules, energy levels of 135–137molecules properties 134–135monochromatic light 141monochromator 143, 144monoclonal antibodies 99optimized, constructs with effector

functions for therapeuticapplication 102–105

monoisotopic mass 987monolithic polymer 288monomer–dimer equilibrium 413monomeric proteins 413monosaccharidebuilding blocks 574sequence 572

3-N-morpholino-1-propane sulfonic acid(MOPS) 697

mouse embryonal fibroblasts(MEFs) 914

mRNA stability 978MS-based proteomics 997MSn-spectra of peptide, measured with

ESI ion trap 374multichannel spectrometers 144

multidimensional HPLC 238design of scheme 240–241fractionation of complex peptide and

protein mixtures by 239purification of peptides and proteins

by 238–239strategies for 239–240

multiphoton fluorescencemicroscopy 198–199

multiple anomalous dispersion(MAD) 540–541

multiple cloning site (MCS) 911multiple isomorphous replacement

(MIR) 538–540multiplicity 135MUSCLE tool 891Mus musculus 942mutated proteins 120Mycobacterium tuberculosis 1009,

1011Mycoplasma genitalium 938m/z values 345, 351, 354

N

N-acetyl-L-glutamate-kinase(NAGK) 414

Naja naja oxiana 863nano-crystals 529nano-HPLC flow rates 375nano-RP-HPLC 650National Center for Biotechnology

Information (NCBI) 875BLAST (Basic Local Alignment Search

Tool) 890yeast Mbp1 protein 889

native chemical ligation (NCL) 1052native proteins 207natural killer cells 104natural light 132nearest neighbor-analysis 827nearest neighbor-assays, methylation

analysis 827–828near-field scanning optical microscopy

(NSOM) 202Neisseria gonorrhoeae 723Neisseria meningitidis 723nephelometry 84Nernst function 151nested PCR 766–767one tube 767

neutral fragment masses 991neutral loss scan 352new antibody techniques 99–102next generation sequencing (NGS) 108,

785application areas 786

N-hydroxysuccinimide-activatedhaptens 737

nick translation 736ninhydrin 304reagent 25

nitrilotriacetic acid 233nitro-blue tetrazolium salt (NBT)coupled optical redox reaction 748

nitrocellulose 652membranes 706

nitrogen laser 331o-nitrophenol (ONP) 915o-nitrophenyl-β-D-galactopyranoside

(ONPG) 915p-nitrophenyl diazopyruvate 126, 127nitroxides 480protonated/unprotonated,

structure 480N6-methyladenine 817N-nitroso-alkylating reagent 865NOESY spectra 459Nomarski, Georges 182non-coding RNAs (ncRNAs) 856non-competitive inhibitors 41non-crystallographic symmetry

(NCS) 540nonionic detergents 20triton X-100 673

nonlinear anisotropic diffusion 513nonpolar gas chromatographic

columns 621nonpolar ligands 227non-protein nitrogen 25non-radioactive detection systems 742non-radioactive labeling 739bioluminescence 748–749biotin system 745–746chemiluminescence 747–748digoxigenin system 744–745dinitrophenol system 746direct detection systems 740–742electrochemiluminescence 748fluorescein:anti-fluorescein (FLUGS)

system 746fluorescence detection 749FRET detection 749indirect detection systems 742–747luminescence detection 747optical detection 747signal-generating reporter group 740in situ detection 749

non-radioactive modifications 733non-radioactive reporter groups 741non-small cell lung cancer (NSCLC) 1041N-terminal sequence analysis 315nuclear magnetic resonance

(NMR) 1026spectroscopy 107, 120, 314,

433–434, 486, 530Bloch equations 436–437limitations 433

Index 1099

nuclear magnetic resonance (NMR)(continued )

nuclear spin and energyquantization 434–435

populations and equilibriummagnetization 436

pulsed Fourier transformationspectroscopy 437–438

relaxation 437speeding-up 463theory 434–438

nuclear-run-on assays 905nuclear-run-on transcription 9065´ nuclease reaction format

(TaqMan) 726coupled amplification/detection 727homogeneous detection systems 727

nucleic acid blotting 704colony/plaque

hybridization 707–708dot/slot-blotting 707membrane, choice of 704–705Northern blotting 706–707Southern blotting 705–706

nucleic acids 705amplification systems 750signal amplification 752–753target amplification 751–752

blotting (see nucleic acid blotting)detection systems 738by hybridization 722non-radioactive systems 739–749radioactive systems 738–739staining methods 738

electrophoresis (see electrophoresis)fragments isolation, purification 729using electroelution 708–709using gelfiltration/reversed phase 708using glass beads 708

in gel matrix 691hybridization 719basic principles of 720heterogeneous systems forqualitative analysis 723

heterogeneous systems forquantitative analysis 723–724

homogeneous systems 725, 729intercalation assay 728–729molecular beacon system 728practice of 721–722in situ assays 729specificity 722–723TaqMan/5´ nuclease amplificationdetection 725–728

isolation of fragments (see nucleic acidfragments, isolation of)

labeling methods 733chemical labeling 737–738enzymatic labeling 735–737

photochemical labelingreactions 737

positions 733–735mixture 700, 721photoactive substances for detection

of 737probes for 729DNA probes 730–731LNA probes 732–733PNA probes 732RNA probes 731–732

restriction analysis 681historical overview 682principle of 681–682

restriction enzymesbiological function 682classification of 682–683isoschizomeres 685recognition sequences 683–685type II 683

staining (see staining methods)stringency, specificity 722–723in vitro restriction/applicationscomplete restriction 685genetic fingerprint 689genomic DNA, restriction analysisof 686–688

incomplete/partial restriction 686methylated bases, detection of 688multiple restriction enzymes,combination of 686

partial restriction 686restriction fragment lengthpolymorphisms(RFLP) 688–690

restriction mapping 686, 687, 688nucleic acid sequence-based amplification

(NASBA) 751, 755, 778nucleic acids, isolation/purification ofalkaline lysis, principle of 672ampicillin-containing media 670carrier 668cetyltrimethylammonium bromide

(CTAB ) 670CsCl density gradient 673cytoplasmic RNAcultivated cells 677tissue/cultivated cells 677–678

determination ofconcentration 668–669

DNA yield after anion exchangepurification 673

double-single-stranded DNAabsorption curves of 668

with ethanol 667eukaryotic low molecular weight

DNA 674eukaryotic viral DNA 675gel filtration 666–667

genomic DNA 669additional steps 670cell membranes/protein degradation,lysis of 669

enzymes/lysis reagents 669phenolic extraction/subsequentethanol precipitation 670

precipitation 670lab-on-a-chip (LOC) system 680low molecular weight DNAanion exchangechromatography 673

bacterial culture 671density gradientcentrifugation 673–674

lysis of bacteria 671–673plasmid from bacteria 670–671

magnetic particles 679–680optical density (OD) 668phenolic purification 665–666photometric determination 668plasmid DNA by CsCl density gradient

centrifugation 674precipitation with ethanol 667–668protein containing

contaminations 665RNA isolation 676poly(A) 678–679

sensitive quantification method 669single-stranded DNA 676double-stranded DNA 676M13 phage DNA 676

small RNA, isolation of 679Tris-HCl or TE (Tris-HCl/

EDTA) 665viral DNA, phage DNA 674–675

Nucleic Acids Research (NAR) 877nucleophiles 125nucleotide sequence 876management, in laboratory 881

nucleotide triphosphate 951numeric aperture (NA) 186, 187

O

object characteristics 501–502, 504n-octyl-β-D-thioglucopyranoside

(OSPG) 673octylglucoside 59, 60off-gel isoelectric focusing, principle

of 266Ogston sieving effect 691Ohm’s law 293oil-immersion objective 187okadaic acid 1045oligodeoxyribonucleotide, comparison

of 963oligonucleotides 738, 793, 960antisense 960, 963

1100 Index

cell culture/animal models 964human β-globin pre-mRNA 961intermolecular triple helix 962mechanisms 960–961RNase H 960–961RNA splicing, changes 961as therapeutics 964–965translation inhibition 961

aptamers, high-affinity RNA/DNA-oligonucleotides 971–974

arrays 731CGE electropherogram of 702fingerprinting 936gapmer 964high-affinity 973interferon response 967intermolecular triple helix 962mechanism and location 960micro-RNA pathway 970nucleotides modifications 962oligosaccharide binding fold (OB

fold) 860positions for nucleotides

modifications 962probes 918disadvantage of 722

ribose, by fluorine2´ position 973

RNA interference, mechanism of 968SELEX-strategy, for RNA aptamers

isolation 972short hairpin RNA (shRNA)vector expression 969

susceptibility to nucleases 962–964synthesis 709, 710, 952phosphoramidites 737

triplex forming oligonucleotides(TFOs) 961–962

uses 959oligosaccharides 572omics 1023, 1024one-dimensional diffusion 76one-dimensional NMR

spectroscopy 437–438chemical shift 4391D experiment 438line width 442scalar coupling 439–442spectral parameters 438–439

online detection systems 798online sample concentration 295–296one buffer stacking system 295two buffer stacking system 295–296

open reading frames (ORFs) 789o-phosphoserine (OPS) 233o-phthaldialdehyde (OPA) 118optical enzymatic detection systems 747optical multichannel analyzers

(OMAs) 144

optical rotation dispersion (ORD) 178optical spectroscopic techniques 131, 132physical principles 132

optical tweezers 856orbital ion trap 349–350, 350–351Orbitrap 350linear ion trap with 354–355

organic radicals 466organic solvents 10ortho-phthaldialdehyde (OPA) 305, 306oscillating dipole moment 172Ouchterlony immunodiffusion

technique 78

P

PacBio RS single molecule real time DNAsequencing 814

paired-end-sequences, alignment of 811paired-end tag sequencing 829pair-end sequencing 810Pancreatic DNase I 827paraffin slices 197parallel reaction monitoring

(PRM) 1012paramagnetic centers 467paramagnetic dipole interaction 119parfocal distance 185partition coefficient 59, 60passive immunoagglutination 72Pauli principle 135PCR. see also bisulfite PCRpeak capacity 241peak dispersion 223peak distance 221peak variances 223peak widths 221pentose phosphate pathway 1024pepsin 207peptide. see also peptide based

quantitative proteome analysisanalysis 329antibodies 99bonds 23, 30, 224hydrolysis of 302

detection of 651ESI-MS spectrum 373fragmentation 562general structure 313monoisotopic masses 989separation and enrichment 649sequences 313

Peptide Atlas projects 1008Peptide Atlas SRM Experimental Library

(PASSEL) 1004peptide based quantitative proteome

analysis 998bottom-up proteomics 998data analysis/interpretation 999

ionization 999mass measurement/fragmentation 999

peptide separation 998proteolysis using trypsin 998

bottom-up proteomic strategies 1000DDA, principle of 1000DIA, principle of 1001SRM, principle of 1000–1001

data dependent analysis (DDA)challenge 1003principle/intended use 1002strength/weaknesses 1002typical applications 1003

extensions 1012MSE 1013MSX 1013parallel reaction monitoring(PRM) 1012

precursor acquisition independentfrom ion count(PAcIFIC) 1012–1013

peptide quantification 1001–1002proteome, complexity of 1000selected reaction monitoring

(SRM) 1003analysis software 1007–1009clinical studies 1010data analysis 1007eukaryotic modelorganisms 1009–1010

identification 1005–1006method 1004–1005microbial 1009principle of 1003–1004quantification 1006–1007strength/weaknesses 1009typical applications 1009

SWATH-MSprinciple/intended use 1010–1011strength/weaknesses 1011typical applications 1011–1012

peptide libraries, analytics of 567–569approach to identifying 568characterization 569divide–couple–combine method 567Edman degradation 567

peptide nucleic acid (PNA) 730oligomers 732

peptide synthesis 555–560common side products and side

reactions during 560approach 568characterization by ESI massspectrometry 569

divide–couple–combinemethod 567

Edman degradation 567Fmoc- or Boc-strategy 557

Index 1101

peptide synthesis (continued )and identification 555important reaction mechanisms

in 558principles 555structure and abbreviation of selected

agents used in 557synthesis on solid support 556

peptide synthesizer 559peptidomeanalysis of 1029collagenases 1029

peptidomics 1023, 1028–1029peroxidase–anti-peroxidase (PAP)

complex 87Petran, Mojmir 182PFGE gels 699Pfu DNA polymerases 759phagocytosis 64, 105phase contrast condenser 185phase contrast microscopy 187phase contrast objective 185phase diagram of aqueous protein

solution 532phase transitions 53, 54phenol/chloroform/isoamyl alcohol

(PCIA) 665phenolic extraction 666phenotypic/biochemical markers 931phenylalanine 225phenyl isothiocyanate (PITC) 306, 654phenylthiocarbamoyl (PTC)-

peptide 315phosphatase/deacetylase-treated

sample 652phosphatidylcholines 50, 641phosphatidylethanolamines 641phosphodiester bonds 856phospholipases 615phospholipid dimyristoyl-

phosphatidylcholine 59phosphomethylene-L-phenylalanine

(Pmp) 1054phosphopeptides 650phosphoric acid (H3PO4) 650phosphorothioates, disadvantages

of 962phosphorylated proteins 650analysis of 1054detection of 651separation and enrichment 649

phosphorylation 224, 329, 369, 561,645–646, 979

phosphoserine 648phosphotyrosine-containing proteins,

generic detection of 652photoactivatable groups 127photoactivated localization microscopy

(PALM) 201, 486

photoaffinity labeling 121, 123photobleaching 199photodetectors 133photolabeling peptides, with

benzophenone 127photolysis 125, 126caged probe 1051

photometric measurement 142,143–144

frequent errors in 143main sources of error in 142principle of 132–133with circular polarized light 133with linear polarized light 132

photon emission 915photon energies 133photon-induced dissociation (PID,

IRMPD) 360photoreceptors 133phototoxicity 200Phred quality 808pH sensors 429pH values 561phycoerythrins 741physical-chemical systems 35pico-Newtons (pN) 855piezoelectric quartz crystals (PQC) 429pigment–protein complexes 140, 152pI value 561pixel protein profiling (PPP) 1062, 1063plan achromatic objectives 185plan apochromatic objectives 185planar/bead-based arrays 1034planar invitrogen microarray 1038planar protein microarray 1033plasma proteins 981dynamic range 981

plasma proteome analyses 981plasmid vector 788ColE1 origin 671copy numbers 671

plasminogen 995plate number 222, 223, 235point spread function (psf) 185polarimeter 178polarity 224, 225polarization 132microscopy 188plane 178

polarized light 132linear dichroism 175–178methods using 175

polyacrylamide gel electrophoresis(PAGE) 711, 901

polyacrylamide gels 244, 694, 704advantages of 260separation ofoligonucleotides 696range 695

structure 250vs. agarose 694

polycystic kidneys 767polydimethylsiloxane (PDMS) 277polyethersulfone 16poly(ethylene glycol) (PEG) 675poly(ethylene oxide) (PEO) 701polyketides (PK) 614poly-L-lysine 180polymerase chain reaction (PCR) 721,

725, 751, 755, 918, 935, 950alternative amplification

procedures 777nucleic acid sequence-basedamplification (NASBA) 777

transcription-mediated amplification(TMA) 777

Alu PCR 770amplification 819avian myeloblastosis virus (AMV)RTase 761

enzymes 761Moloney murine leukemia virusRTase 761

primers 762procedure 762RNA (RT-PCR) 761in single reaction tubes 762Tth DNA polymerase 762in two reaction tubes 762

applications 772genetic defects, detectionof 773–776

human genome project 776–777infectious diseases, detectionof 772–773

branched DNA amplification (bDNA)method 782

contamination problems 770avoiding 770–771decontamination 771–772

digital PCR 769DNA amplification 758, 763additives 763buffer 759cycles 758–759enzyme 759hot start PCR 763magnesium ions 763nucleotides 759primers 759–760probe preparation 758RNA amplification 763template 763templates 760

for DNA brand marking 794DOP PCR 770helicase-dependent amplification

(HDA) 777–779

1102 Index

instruments 756–757inverse PCR 769ligase chain reaction (LCR) 779–780master mixes 759mutagenesis techniques 870optimization of reaction 763polymerization of DNA 756possibilities of 755–756PRINS PCR 770prospects of 782Qβ amplification 780–781quantitative PCR 763competitive (RT) 765–766external standard 764–765internal standardization 765

RACE PCR 769repair chain reaction (RCR) 780RT and Taq DNA polymerase 762schematic of 757special techniques 766asymmetric PCR 767cycle sequencing 768degenerate primers, use of 767homogeneous detectionprocedures 768–769

multiplex PCR 767–768nested PCR 766–767quantitative amplificationprocedures 769

in situ PCR 769in vitro mutagenesis 768

strand displacement amplification(SDA) 777

temperature/time profile of 758typical course 764vectorette PCR 769

polymerization 251poly(methyl methacrylate)

(PMMA) 277, 680polymorphic markers 927, 931polymorphic sequence tagged sites

927Polyoma/SV40 nucleic acids 675polypeptide 147poly(vinylidene fluoride) (PVDF) 16,

652membrane 984membranes 273

polyvinylpyrrolidone (PVP) 14, 701pore size 251, 255porosity 288gradient gels 254–255

positional candidate gene approach 939positional cloning 938position-specific scoring matrix

(PSSM) 883post-translational modification

(PTM) 314, 315, 369, 645, 647,648, 659, 998

amino acids, localization/identification 653–654

analysis, based on 648future perspective 661quantitative analysis of 659–660

potential energy 35power of combinatorial molecular

discrimination (PCMD) 1061,1063

33P phosphates 733precipitation 3, 9, 17, 64of nucleic acids 10–11using organic solvents 10using trichloroacetic acid 10

precursor acquisition independent fromion count (PAcIFIC) 1012

precursor ion analyzes 352prenols (PR) 614preparative immunoprecipitation 81–82preparative techniques 263electroelution from gels 263–264isoelectric focusing 265between isoelectric membranes,principle of 265

preparative IEF between isoelectricmembranes 265–266

preparative zone electrophoresis 264preparative zone electrophoresis 264pressure perturbation calorimetry

(PPC) 61primers 819extension assay, principle of 902oligo(dT) primers 760for RT-PCR 762secondary structures of 760types of 759

principal component analysis(PCA) 506–508, 1031

approach of 506factorial map 508independent structures 507mathematical procedure 507motivation for 508of prealigned projections of the protein

complex 508real EM data 508real EM images, number of

pixels 507representation of two-pixel images in

coordinate systems 507sensitivity of 508

product ion analysis 352prompt and metastable decay

(ISD, PSD) 358–360spectrum of angiotensin 372

Prosite Motif PS00029 882prosthetic groups 23protease 588protease inhibitors 8

protein aggregation 49protein based quantitative proteome

analysis 982intact protein mass spectrometry,

concepts 987–997closing remarks/perspective 996–997data analysis 991–995high-throughput top-downproteomics 995

mass spectrometry, to measure intactproteins 990–991

top-down proteomicsusing intact protein massspectrometry 987

using isotope labels 986–987two-dimensional differential gel

electrophoresis (2DDIGE) 986two-dimensional-gel-based

proteomics 982peptide fragments, analysisof 985–986

proteins, imaging/quantification 983–985

proteins separation 982–983sample preparation 982

protein co-compartmentalizationmachine 1058

protein complexes 530protein crosslinking 121protein crystallization using the hanging

drop method 532Protein Data Bank (PDB) 543protein degradation 978protein determination 23method for 24staining methods for 26

protein–DNA interactions 951protein dynamics, determination

of 463–464protein equivalent of a genome 977protein expression 978protein folding/misfolding 464protein fragmentation 990, 992, 993protein functional groups, chemical

modification of 108–116acylation 109–110amidination 110arginine residues 113caged compounds 111cysteine residues 112glutamate and aspartate

residues 112–113histidine residues 116–117lysine residues 108methionine residues 114–115reaction with isothiocyanate 111reductive alkylation 110–111tryptophan residues 114tyrosine residues 113–114

Index 1103

protein functionsbiologically active molecules for

modulation 1044switching off 1044

protein glycosylation 572, 579analysis of 581

protein imaging 1068protein interactions 1036protein–ligand interactions 299protein localization 6protein mass spectrometry 987protein microarray applications,

schematic representation 1037protein microarrays 1037protein–nucleic acid complexesby gel electrophoretic methods 836molecular beacons 853

protein–nucleic acids interactions 831dissociation constants, determination

of 839–840DNA footprint analysis 841chemical nucleases 849–850chemical reagents formodification 846–848

genome-wide 850–851hydrolysis methods 844–845interference conditions 848–849labeling 843primer extension reaction 843–844

DNA–protein complex dynamics,analysis of 840–841

filter binding 836gel electrophoresis, background to

retardation 836–838genetic methodsaptamers/Selex procedure 869directed mutations, within bindingdomains 870

tri-hybrid method 868–869physical analysis methodsfluorescence correlationspectroscopy (FCS) 856

fluorescence methods 851fluorescence resonance energytransfer (FRET) 852–853

fluorophores procedures 851–852labeling procedures 851–852molecular beacons 853optical tweezers 855scanning force microscopy(SFM) 854–855

surface plasmon resonance(SPR) 853–854

RNA interactions (see RNA–proteininteractions)

proteinogenic amino acids 876protein profilesdisease specific 100-dimensional

discovery 1062

protein-protein interactions (PPIs) 381,555, 1046

protein purification 23, 219, 315goal of 5

proteinschemical modification 107complexity and individuality of 23complex structures, three-dimensional

reconstruction of 509denaturation of 209alkylation of cysteine residues 210cleavage of disulfide bonds 209cysteine residues, chemicalmodification of 210

disulfide bonds andalkylation 209–210

2D gel 984immunogenicity of 70localization of 1033peptides, separation methods of 4properties of 3size 3splicing, conditional 1054–1055staining 983

ProteinScape 1019protein structure, determination 457,

462high molecular weight systems and

membrane proteinstructure and dynamics of 465–466

in-cell NMR spectroscopy 466intrinsically disordered

proteins 464–465NMR spectroscopy 462–463NOE signal intensity and respective

proton distance, relationshipbetween 457

protein dynamics,determination 463–464

protein folding and misfolding 464protein–ligand complexesthermodynamics and kineticsof 464

residual dipolar couplings(RDCs) 458

secondary structure, determinationof 458–461

structure calculation, constraintsfor 457–458

tertiary structure, calculationof 461–462

distance geometry method 461root-mean-square deviation(RMSD) 462

simulated annealing 461protein toponome, concept

of 1058–1059protein transfer 88proteoforms 981

proteolytic enzymes 207–208cleavageon membranes 208in SDS-polyacrylamidegels 208–209

in solution 208strategy 208

proteome analysis 108, 610–611, 977based diagnostics 956coverage 1012general aspects 977protein based quantitative analysis (see

protein based quantitativeproteome analysis)

ProteomeXchange 1010proteomic databases 611sample preparation 980–982SDS gel electrophoresis 977starting conditions/project

planning 979–980prozone effect 72protospacer adjacent motif (PAM) 974PTEN knockout 1010pulsed electron double resonance

(PELDOR) 478pulsed EPR experiments 473basics 474electron nuclear double resonance

(ENDOR) 477–478electron spin echo envelope modulation

(ESEEM) 475–476hyperfine sublevel correlationexperiment

(HYSCORE) 476–477pulsed electron double resonance

(PELDOR) 478relaxation 474spin echoes 474–475

pulsed-field gel electrophoresis(PFGE) 698, 934

pulsed laser beam 1064pulsed liquid sequencer 321pure protein solutions 24purification techniques 4, 6Pwo DNA polymerases 759pyridoxal-5´-phosphate 111pyrimidines, 5,6-double bonds of 846pyrophosphorolysis 793pyrosequencing 807, 820

Q

quadrupole analyzer 345–348quadrupole-TOF (Q-TOF) 354analyzers 377

quantitative determination, by stainingtests 25–26

quantitative immunoprecipitation 73quantitative structure–retention

relationships (QSRRs) 241quantitative trait loci (QTLs) 940

1104 Index

quantum dotsas fluorescence labels 159–160labeling with 192

quasi-isothermal conditions 48

R

RabGDP-dissociation inhibitor(RabGDI) 1053

Rab-GTPase Ypt1 1052radioactive detection methods 652radioactive labeling 31–33, 109, 733of DNA sequencing 797exchange positions 735

radioactive methods 651radioactive nucleic acidsdirect autoradiography 739fluid emulsionsfor cytological/cytogenetic in situapplications 739

fluorography 739indirect autoradiography, with

intensifier screens 739pre-exposed X-ray film, for direct

autoradiography/fluorography 739

radioimmunoassays (RIA) 31, 82–83radioisotopes 739radiolabeling 32Raman spectroscopy 171, 1026principles 171–172Raman experiments 172–173

Raster image correlation spectroscopy(RICS) 203

Rayleigh–Gans–Debye scattering 141Rayleigh scattering 142Rd1-SP-adapter ligation 812Reactome 1024reagents for introducing

fluorophores 117real-time RT-PCR (RT-qPCR) 904, 905quantification of gene expression 904

REases 683type-II restriction enzymes 684

recombinant antibody 100recombinant DNA technologies 120recombinant glycoproteins 571recombinant proteins 7recombinant retroviruses 395recombinaseA(recA-), strains deficient

of 671recombination fraction 929red-green-blue (RGB) images 1065reductive alkylation 110–111RefSeq 890regular expression functions 882regularly arrayed macromolecular

complexes, three-dimensionalreconstruction 511–512

relative centrifugal force (RCF) 12relative molecular mass 4relative resolution map (RRM) 236Renilla luciferase 749reporter gene assay 1047reporter gene vectorcis-acting sequencesprinciple of mapping 912

resolution 185capacity 342optimization 224power of isoelectric focusing 260range, different methods for 529

resonance assignment 452heteronuclear 3D spectra, analysis

of 454selective amino acid labeling 454sequential assignmentof homonuclear spectra 452–453from triple-resonancespectra 454–457

resonance methods 134resonance Raman

spectroscopy 173–174restricted access materials (RAMs) 238sorbents materials 229

restriction analysis, methylation sensitiveenzymes/insensitiveisoschizomers 824

restriction enzymes 682cleavage 685

restriction fragment lengthpolymorphisms (RFLPs) 681,927, 928

retention 228factor 221, 223, 235times 220, 221volume 221

reveal bacteria 671reversed-phase chromatography

(RPC) 16–17, 240reversed-phase HPLC 21reverse immunoagglutination 72reverse phase protein microarrays 1038reverse transcriptases (RTases) 761,

896, 901RNA-dependentDNApolymerase 901

Rev protein 859rhodamine-tagged

phosphoramidites 851Rhodobacter capsulatus 157rhodopsins 149, 176ribonuclease-protection assay

(RPA) 898–901, 900ribonucleases (RNases) 959ribonucleoprotein (RNP) 856domain 859

ribose by fluorine2´ position of 973

ribosomal RNA 904ribosomes 530transfer-RNA (tRNA) molecules 947

ribozymes 479, 959, 965, 967catalytic cycle of 966discovery/classification 965–966structure of 966use of 966–967

RNAaptamers, SELEX-strategy 972characteristic structural elements 858DNA, helical grooves between 857mimic fragments 765moleculesgel electrophoresis, two-dimensional 701

splicing, analyzing 947RNA-binding motifs,

characteristic 859–860RNA-binding proteins 859RNA-dependent DNA polymerases 761RNA-DNA hybrid molecules 896, 901RNA/DNA sequencescloning/PCR amplification 788electrophoresis 788error correction/sequence data

analysis 789nucleic acid, isolation/purification

of 788purification 788reconstitution 788–789

RNA electrophoresis 697RNA-induced silencing complex

(RISC) 967RNA interference (RNAi) 959,

967–971, 968, 969basics of 967–968mediated by expression

vectors 968–969uses of 969–970

RNA isolation 679RNAi-triggered cellular processes 968RNAi-triggered knockdown 955RNA-modifying reagentsreagents for 864structural formula 864

RNA polymerases 899, 905, 908analysis of binding 855RNApolymerases transcribe 908

RNA-protein complexes 866analysis of 860chemical modification 863–866chemical crosslinking 866–867CMCT (1-Cyclohexyl-3-(2-

morpholinoethyl)carbodiimidemetho-p-toluolsulfonate)864–865

customary RNases 862–863diethyl pyrocarbonate (DEPC) 864

Index 1105

RNA-protein complexes (continued )dimethyl sulfate (DMS) 864ENU (ethylnitrosourea) 865Fe-BABE

(Fe 1-(p-bromoacetamidobenzyl)ethylenediaminetetraaceticacid) 865

hydroxyl radicals 865in-line probing 865kethoxal (α-keto-β-ethoxy-

butyraldehyde) 863–864labeling methods 861–862limited enzymatic hydrolyses 861nuclease S1 863photoreactive nucleotides,

incorporation of 867primer extension analysis 862RNase CL3 863RNase CVE 863RNase T1 862RNase T2 863RNase U2 862selective 2´-hydroxyl acylation

analyzed by primer extension(SHAPE) analysis865–866

transcription start sites (TSS),genome-wide identificationof 867–868

RNA-protein interactions 857dynamics of 857–859functional diversity 856–857secondary structure parameters/unusual

base pairs 857RNA–protein recognitiontri-hybrid system for the in vivo

characterization of 868RNA quantification, by Northern

blot 903RNA-RNA hybrid molecules 899RNase H activity 777, 961RNase inhibitors 676RNAse protection assays 761RNASeq 813, 946RNases, specificities of 860, 861RNA transcriptsdot- and slot-blot analysis 903–904Northern blot 902–903nuclease S1 analysis 896, 897quantitative 896reaction principle 896RNA 5´/3´ ends 897–898

overview 895–896primer extension assay 901–902reporter gene expression 914β-galactosidase (β-Gal)915

chloramphenicol acetyltransferase(CAT) assay 914

green fluorescent protein(GFP) 915

luciferase assay 915transcripts from transfectedcells 915

reverse transcription polymerase chainreaction (RT-PCR) 904–905

ribonuclease-protection assay(RPA) 898–901

in vitro transcription, in cell-freeextracts 907

additional techniques toanalyze 911

G-less cassette 908–909run-off transcriptionassays 909–910

template DNA/detection 908transcription assay 907–908transcription-competent cell extracts/protein fractions, generationof 908

in vivo analysisnascent RNA labeling with 5-fluoro-uridine (FUrd) 906–907

nuclear-run-on assay 905–906ROBETTA server 893robot-assisted microdispension

system 610robotic system 50rocket immunoelectrophoresis 80–81Rohrer, Heinrich 486root-mean-square deviation

(RMSD) 462Rosetta program 893rotors, for centrifugation 11Rous sarcoma virus (RSV) 960Royal Society of London 181RP-LC-ESI-MS proteolytic digest of

protein 377RT-PCR techniques 904schematic portrayal 761

run-off transcription reaction 910Ruska, Ernst 486

S

Saccharomyces cerevisiae 785, 938,942, 947, 1009, 1037

salts removal 15sample concentration 295sample preparation 195, 249, 332–335creation of paraffin slices 197embedding 196–197fixation 196frozen slices 197creation of frozen slices (rapidslices) 197

embedding 197sealing 197

isolated cells 195–196paraffin samples 196proteome analysis 22tissue biopsies 196

Sanger method 785, 786, 805Sanger’s reagent 313scanning calorimetry 47scanning cysteine accessibility method

(SCAM) 120scanning electron microscope

(SEM) 486scanning force microscopy

(SFM) 854–855scanning ion conductance microscope

(SICM) 519scanning microprobe MALDI

(SMALDI) 1065, 1068mouse spinal cord 1070mouse urinary bladder tissue

section 1069scanning near-field infrared microscopy

(SNIM) 486scanning near-field optical microscopy

(SNOM) 486, 519scanning probe microscopies

(SPM) 486scanning tunneling microscope

(STM) 486scattering artefacts, correction for 142scFv-Antibodies 100Schiff base 111, 149Schlack–Kumpf degradation 326Schleiden, Matthias J. 182Schwann, Theodor 182secondary electron multiplier (SEV)

356channel electron multiplier 356constructions with discrete

dynodes 356microchannel plates 356

secondary ion mass spectrometry(SIMS) 1064

ion imaging mode 1064secretory proteins 886sedimentation coefficient 12, 412sedimentation–diffusion

equilibrium 415selected or multiple reaction monitoring

(S/MRM) 1000mass spectrometer 1004quantification 1002

selective 2´-hydroxyl acylationanalyzed by primer extension(SHAPE)

reagents 866selectivity 221, 239selenium 25Selex procedure 869separated proteins zones 251

1106 Index

detection and quantification 251–252imaging 252–253

separation efficiency 4sequence composition 882sequence data analysis 875abstraction for biomolecules 876and bioinformatics 875–876homology based methodsbasic local alignment search tool(BLAST) 890

identity 887–888optimal sequencealignment 888–890

PSI-BLAST algorithm 890–891similarity 887–888threshold 891

internet databases/services 877data contents/file format 879–880nucleotide sequence management, inlaboratory 881

sequence retrieval, from publicdatabases 878–879

multiple alignment/consensussequences 891–892

procedures 893structure prediction 892–893

sequence logo 883, 886sequence patterns 882–886coding regions, identification

of 885–886protein localization 886transcription factor binding

sites 884–885sequence tagged sites (STSs) 776, 927physical markers 927screening 936

serial femtosecond crystallography(SFX) 550

serum amyloid A (SAA) 96short hairpin (SH)groups modification 112with p-chloromercury benzoate,

analysis 112short-hairpin RNA (shRNA) 953, 968vector expression 969

shotgun method 786sickle cell anemia 688signal amplification 751branch structures 752coupled signal cascades 753cyclic ADH 753enzyme catalysis 752–753

signal-to-noise ratio 144, 504–506averaging single particles 505–506correlation averaging 504–505filter approaches for crystal data 504filtering in Fourier space 504

silica coated magnetic beads 679silver staining 251, 252, 704

simplified coordinate systemvisualization of experimental

data 1031single beam photometer 142single crystals, of amylase C 533single ion monitoring (SIM) scan

mode 348single isomorphous replacement

(SIR) 538–540single molecule spectroscopy 174–175single nucleotide polymorphisms

(SNPs) 927, 940identification of 948microarray-based 948polymerase extension reaction 949SNV analysis 940

single nucleotide variants (SNVs) 927,940

single particleanalysis 516three-dimensional

reconstruction 509–511single reaction monitoring (SRM)and MRM-analysis 352–353SRMAtlas project 1008SRMCollider 1005, 1009

single-strand conformationalpolymorphism (SSCP) 695,775–776

single-stranded binding (SSB) 742single-stranded DNA(ssDNA) 775single-stranded RNA probes 731singlet oxygen generator (SOG) 409singlet oxygen sensitizer 409singlet oxygen triplet energy transfer

(STET) 409size exclusion chromatography

(SEC) 240Skyline software 1008slot-blotting 707small angle X-ray scattering

(SAXS) 529, 543–544data analysis 547de novo structuredetermination 547–548

method developments 549machine setup 544–545theory 545–547

SMART server domain 884snake venom phosphodiesterase 827sodium bisulfite catalyzes 819sodium dodecyl sulfate (SDS)electrophoresis, for low molecular

weight peptides 258PAGE, analytical

ultracentrifugation 409polyacrylamide gel

electrophoresis 257–258sodium saline concentration (SSC) 723

software tools 984solid phase sequencer 320soluble macromolecules 529somatic cells 925spatial frequency 499spectral absorption coefficients 25spectral unmixing 204spectroscopic methods 28–29fluorescence method 31measurements in UV range 29–30protein determination 29spectral range 134

spectroscopy. see atomic forcemicroscope (AFM)

sphingolipids (SP) 614sphingomyelins 641spin label 119reagents for 120

splice acceptor (SA) 938splice donor (SD) sites 938splitting scheme, for unpaired

electron 469spot patterns, phosphorylation status of

proteins 652(S)SIM ((saturated) structured

illumination microscopy) 201stable isotope labeling 1014by amino acids in cell culture

(SILAC) 1015bottom-up proteomicsisobaric labeling 1020–1021non-isobaric labeling 101918O labeling 1019reagents 1019–1020

peptide standards 1006in quantitative proteomics 1013top-down proteomics 1013chemical stable isotopelabeling 1016

isotope-coded affinity tag method(ICAT) 1016–1018

isotope coded protein label(ICPL) 1018–1019

metabolic labeling 1014–1015stable isotope labeling by aminoacids in cell culture(SILAC) 1015–1016

staining methodscharacteristics 983fluorescent dyes 702DNA geometry, influence 703ethidium bromide (3,8-diamino-5-ethyl-6-phenylphenanthridiniumbromide) 702–703

fluorescent dyes 703–704silver staining 704

standard free energy 61Staphylococcus aureus 768stationary phase 221, 222, 618

Index 1107

stereoisomers 572sterols (ST) 614stimulated emission depletion

(STED) 201microscopy 486

stochastic optical reconstructionmicroscopy (STORM) 201

Stokes radius 254strand displacement amplification

(SDA) 779Streptomyces avidinii 746strong cation exchange (SCX)

chromatography 649, 650structural proteins 254substance libraries, source of 1045N-succinimidyl-3 [4-hydroxyphenyl]

propionate 109D-sugars series 572composition starting from

D-glyceraldehyde 572L-sugars series 574sulfonyl chloride 116sulfuric acid 25surface plasmon fluorescence

spectroscopy (SPFS) 402surface plasmon resonance

(SPR) 853–854Biacore techniquemeasurement of antibody binding toantigen using 94

measuring device, principle of 854spectroscopy 400–402

surface tension 227, 231Svedberg equation 412, 415Svedberg, Theodor 244Svensson–Rilbe’s concept of “natural”

pH gradients 244SWATHAtlas 1010SWATH-MS 1011SYBR Green 728, 905synthetic peptidescharacterization/identity of 562–564peptide nucleic acids (PNAs) 732purity of 561–562structure, characterization

of 564–567systematic evolution of ligands by

exponential enrichment(SELEX) 972, 973

systems biology, hypotheses/knowledgecircular system 1030

454-system workflow 808

T

tandem affinity purification(TAP) 394–395, 395–397

advantages 395limitations 397

limitations of 397mass spectrometric analysis 397purification 395–397retroviral transduction 395tagging and purification of protein

complexes 394tandem mass spectrometryphosphorylated and acetylated amino

acids, localization of 654–659tandem-TOF (TOF-TOF) 353–354TaqDNA polymerases 730, 736, 758, 759TaqMan® 756, 759PCR 765probes 725, 905

target amplification 751elongation 751transcription 751in vivo amplification 751

targeted proteomics 1004target protein, temporal control 1051TATA binding protein (TBP) 835TAT protein 481t-butyl trifluoroacetate 559T-cells 96T-Coffee 892T7 DNA polymerase-catalyzed

sequencing reaction 791, 792T-effector cells (TE) 105temperature gradient gel electrophoresis

(TGGE) 701, 840, 841terminator exonuclease (TEX) 868test strips 420test system set-up 41–42controls 45detection system 42pH value 43physiological function, analysis of 42selecting buffer substanceand ionic strength 43–44

selecting substrates 42substrate concentration 44temperature 44time dependence 43

TET enzymes 817tetrabutylammonium bromide

(TBAB) 711Tetrahymena thermophila 965, 1068N,N,N´,N´-tetramethylethylenediamine

(TEMED) 292tetramethylrhodamine (TAMRA) 749tetramethylrhodamine

isothiocyanate 116tet-repressor (TetR) 1051therapeutic glycoproteins 571thermal degradation 25thermal denaturation 61DNA species 720

thermal shift assay 531thermograms 49, 51

thermosensors 49thermostable DNA polymerases 795thin-layer chromatography (TLC) 561,

618, 696, 911, 914thiols 126three-dimensional electron

microscopy 508three-dimensional NMR

spectroscopy 449HCCH-TOCSY and HCCH-COSY

experiments 449–450HNCA experiment 451–452NOESY-HSQC and TOCSY-HSQC

experiments 449nomenclature of triple-resonance

experiments 451triple-resonance

experiments 450–451threonine 648thymine/cytosine-specific fission

reaction 803thyroid stimulating hormone (TSH) 1035time-of-flight analysis 345time-resolved fluorescence (TRF) 749time-resolved spectroscopy 144–147TiO2 coated magnetic particles 649titer 73titration curve 60analysis 263

TMHMM 886TOPCONS 886toponomecell/tissue, molecular networks

of 1058defined 1057imaging cycler microscopy (ICM)antibody based 1057

map, structural representation 1058reading technology, fundament

of 1059–1063theory, schematic illustration 1060

toponomics analysis 1057biological systems 1057co-compartmentalization and

topological associationrules 1058

total internal reflection fluorescencemicroscopy (TIRFM) 160, 202

total ion current (TIC)chromatogram 713

T7 phage DNA polymerases 845T4-polynucleotide kinase (PNK) 901phosphorylates 809

transcriptional profiling analysis 946transcription factors 5transcription-mediated amplification

(TMA) 751, 755transcription start site (TSS) 907, 909transfer-RNA (tRNA) molecules 947

1108 Index

transglutaminase 119translocation 920transmission electron microscope

(TEM) 487–488approaches to preparation 488labeling of proteins 492metal coating byevaporation 491–492

native samples in ice 488–490negative staining 490

beam path 487images of vitrified lipid vesicles 498instrumentation 487–488object holder, grids, and plunger for

biological cryosamples 488phase contrast 495resolution 492–493

transmitted fluorescencemicroscope 188

transposon-mediated DNAsequencing 788

1,4,7-triazacyclononane 233tributylphosphine 209trichloroacetic acid (TCA) 10precipitations 738

triethylamine (TEA) 711triethylammonium acetate (TEAA) 711trifluoroacetates 561trifluoroacetic acid (TFA) 228, 333,

558, 560trifluoromethyl

diazirinobenzoyllysine 1291-trifluoromethyl-1-

phenyldiazirine 126trigonometric operation 501triple-quadrupole (triple-quad) 351–352triplex forming oligonucleotides

(TFOs) 961tripropylamine (TPA) 742tris acetate (TAE) 692tris borate (TBE) 692tris-buffers 863tris-(carboxymethyl)ethylene-diamine 233Triticum aestivum 785Trp1 gene 382trypsin 588, 985, 1019tryptophan 225, 303Tth DNA polymerases 759tumor tissue, differential methylation

levels 826T values 258twin supercoiled domain model 834two-dimensional NMR

spectroscopy 443COSY spectrum 4452D experiment, general scheme

of 443–445heteronuclear NMR

experiments 446–447

homonuclear 2D NMR experimentsof proteins 446

HSQC – heteronuclear single quantumcoherence 447–449

NOESY spectrum 445–446TOCSY spectrum 445

two-hybrid system 381AD fusion proteins and cDNA

libraries 385–386bacterial two-hybrid system (BACTH

or B2H) 389, 391bait proteins, used in Y2H screen 385biochemical and functional analysis of

interactions 393biological relevance 394computational analysis of interactiondata 394

independent verification 393localization 394protein domains and motifs 394

carrying out Y2H screen 386–391construction of bait and prey

proteins 382–385elements of 382modifications 392and extensions oftechnology 391–393

principle of 381–382two-state model, curves 52tyrosine 33, 225, 648

U

ubiquitinmonoisotopic and average m/z

values 990proteoforms, theoretical trypsin

cleavage sites 994top-down mass spectrum 988

ultrafiltration 16, 17ultrahigh pressure liquid chromatography

(UHPLC) 302ultraviolet (UV) 133HPLC chromatogram 717spectroscopy 220, 224UV-diode array detection

(UV-DAD) 224VIS/NIR spectroscopy 146chlorophylls 152–153chromoproteins 147–147cytochromes 149–151metalloproteins 151–152principles 146–147rhodopsins 148–149

uniaxial orientation samples 177UniProt 879unmethylated bases, chemical

modifications 818Ustilago sphaerogena 862

V

Van-Deemter-Knox plots 222, 223van’t Hoff equation 61van’t Hoff transition enthalpy 52V-genes 101vibrational modes 169of peptide bond 169

vibration cell mills 8virtual image 185viscosity 247vitamin A 638vitamin D 638–639vitamin E 640vitamin H 746vitamin K 640von Helmholtz, Hermann 493

W

Watson–Crick base pairing 832, 857,861, 862, 962, 971

Watson–Crick hydrogen bonding 961wavelength 538interference of light 187

wavenumbers 133wave-particle dualism 133WebMOTIF 884Web server based modeling 893Western blot analysis 88, 89, 252,

652autoradiograph-based after

2D-PAGE 652whole protein molecules 1038Wiki pathways 1024Wilkins, Marc 977Wolff-rearrangement 126

X

X-chromosome 951inactivation 817

Xenopus laevis 466XML/JSON 879Xq28, candidate genes 932x-ray crystallography 107, 120, 131,

314, 486, 529, 530, 567crystallization 531–538model building and structure

refinement 542–543phase problem 538–542

x-ray diffractionbasics of 536image of crystal 537

x-ray free electron LASER (XFEL) 549detection and analysis 550machine setup and theory 549–550principle 549samples 550

Index 1109

Y

yeast artificial chromosome (YAC)libraries 935

yeast Mbp1 transcription factors 879Y2H interactions 399Y2H screen 386–391Y2H system 381limits 390

Z

Z-DNA sequences 956Zeeman splitting 468Zernike, Frits 182, 485zero mode waveguide (ZMW) 814zinc-finger proteins 835zirconium oxide 649zonal centrifugation 13–14

zone band broadening 223zone electrophoresis 253–254,

278zwitterionic detergents

20zwitterionic/non-ionic detergents

982zymogens 207activation 208

1110 Index