© 2002 American Chemical Society

ChemicalM odificationsand M SThe historically accepted top-ology of the glycine receptor(GlyR), a major inhibitoryneurotransmitter receptor inthe mammalian spinal cordand lower brain, includes alarge N-terminal domain teth-ered to four transmembraneα-helices, denoted M1–M4.However, recent experimentalresults have not supported thepresence of four such α-helices, and some data havespecifically pointed to M1 as,potentially, not being entirelyα-helical. These findings havegenerated interest in refiningthe structural understandingof the GlyR receptor.

To further examine GlyRtopology, John Leite andMichael Cascio of the Uni-

versity of Pittsburgh chemi-cally modified the proteinand performed mass spectralanalysis (Biochemistry 2002,41, 6140–6148). Specifically,they used tetranitromethane(TNM) to selectively nitratetyrosine residues. They fol-lowed this with proteolytic

digestion and MS, in whichmass-shifted (+45 Da) spectrasignals indicated tyrosinenitration. Because TNM onlyreacts with solvent-accessibletyrosine, the spectra revealthe receptor’s topology.

By running TNM-treatedsamples in parallel to un-treated samples through themass spectrometer, Leite andCascio identified 6 of the 16GlyR tyrosine residues asbeing modified to o-nitro-tyrosine. Four of these resi-dues were located within theN-terminal domain of theprotein. The researchers tookadvantage of a recent high-resolution structure attainedfor an acetylcholine bindingprotein (AchBP), which car-ried a sequence highlyhomologous to the GlyR

N-terminal domain, to evalu-ate the quality of the tyrosineaccessibility method. Se-quence alignments betweenthe two proteins showed thatthe four AchBP residuescomparable to the modifiedGlyR tyrosines were solvent-accessible, while others inthis region were not, cor-roborating the modificationresults.

The remaining two mod-ified residues were locatedwithin the putative trans-membrane M1 region. Butif M1 did fold into a pureα-helix, the two tyrosineswould be well within the inte-rior of the lipid bilayer, pre-cluding them from TNMmodification. The modifica-tion results thus suggest thatM1 does not, in fact, fold in

currents

Optimizing CE AnalysisCompared to 2-DE, capillary electrophoresis (CE) offers short-er protein analysis times, requires smaller samples and fewerreagents, and is readily coupled to MS. Unfortunately, CE ishampered by problems of protein adsorption to the capillarywall and low sensitivity. Mari Tabuchi and Yoshinobu Baba ofthe University of Tokushima (Japan) recently addressed thefirst problem with the use of curdlan, a buffer additive that dis-rupts protein−capillary wall interactions. The second problem,however, has typically required that samples be concentratedusing a modified sample injection method. This method,unfortunately, requires longer sampling capillaries, which re-move the benefit of speed. To get around this problem,Tabuchiand Baba developed a new injection method that uses pressureto concentrate the sample while still using a standard-size cap-illary (Electrophoresis 2002, 23, 1138−1145).

Using a commercial peptide mixture, the researcherscompared the peak resolution and run times of a standardseparation without pressure to similar separations using vari-ous pressure conditions. Tabuchi and Baba found that peakmigration times were reduced when 10 mbar of pressure wasapplied to a sample upon injection and that peak shape wasnot significantly altered. Migration time was further reducedwhen the capillary outlet was free of buffer at the time of pres-surization. This latter result, the researchers explained, wasdue to the freedom of the buffer, and therefore the sample, tomove within the capillary when pressurized.

The researchers then tried to optimize the system by usingvarious pressures for varying lengths of time. The results indi-cated that a 10-mbar pulse of 8 s duration offered optimal

peak resolution, as higher pressures (50 mbar) or shortertimes (2 s) led to peak broadening. Furthermore, the re-searchers found that the addition of curdlan to the buffer atoptimal pressure and time conditions maintained the migra-tion time while improving peak resolution.

The researchers warn, however, that the optimal conditionsmay differ according to the sample content and concentration,

buffer conduc-tivity and vis-cosity, capil-lary length andconditioning,and machinespecifications.Tabuchi andBaba suggestthat the newmethods willenable the useof CE for high-th roughputapplications.

JournalofProteomeResearch •Vol. 1, No. 4, 2002 299

Performing wellunderpressure.When capillary electrophoresis sampleinjection is followed by a burst of pressure, the sample peptides elute morequickly (Method II) than without the pressure (Method I). This effect is fur-ther enhanced by eliminating the buffer from the outlet before sampleinjection (Method III). (Reproduced with permission from Electrophoresis2002,23, 1138−1145. Copyright 2002 John Wiley & Sons Ltd.)

250A

00 1 2 3 4 5 6 7 8 9 10

200B

00 1 2 3 4 5 6 7 8 9 10

150C

00 1 2 3 4 5 6 7 8 9 10

Abso

rptio

n(m

AV)

Method I:sample injection(outlet: buffer)without pressurization

Method II:sample injection(outlet: buffer)water pressurization(outlet: buffer)

Method III:sample injection(outlet: no buffer)water pressurization(outlet: buffer)

1

2

3

4

5

6 78

9

1

2

3

4

5

67

89

1

2

34

5

6

78

9

M odified tyrosines.GlyR residues220−246 are modeled as a trans-membrane α-helix, and tyrosine (Y)residues modified by the tetrani-tromethane are shown as graybeads. (Adapted from Biochemistry2002,41, 6140−6148.)

M220G221

L224I225

Y228I229

S231L232

V235I236

W239I240F242W243

M246

Y222Y223

Q226M227

P230

L233I234

L237S238

S241

I244N245

this manner. The authorspropose that the region maybe composed of multipleβ-sheets or, alternatively, itcould consist of “a novel,unexpected secondary struc-ture.” They are currentlyusing cysteine substitutionmutagenesis coupled to spinlabeling techniques to furtherdefine GlyR M1 topology.

Simulating ProteinFoldingMany human diseases are theresult of genetic mutationsthat lead to errors in proteinfolding, that is, the protein isproduced but is unable toperform its normal function(e.g., sickle cell anemia andhemoglobin). Thus, under-standing which internal andexternal factors influence theway a protein folds to its na-tive state is a pressing issue.Unfortunately, it is difficult tostudy how a protein movesthrough its transition-stateensemble (TSE) from unfold-ed to native structure by con-ventional techniques such asmutagenesis or chemicalcross-linking because the TSEis unstable, transient, andhigh-energy. Computationalmodeling using algorithms,such as molecular dynamics(MD) or Monte Carlo (MC)simulations, however, mayprovide clues to understand-ing the TSE.

Previous MD and MCsimulations of proteinfolding have identifiedtransition-state structuresthat are consistent withexperimental data. Recently,Jörg Gsponer and AmedeaCaflisch of the University ofZurich (Switzerland) com-pared experimental foldingdata to an MD simulation ofthe src SH3 domain TSE(Proc. Natl. Acad. Sci. U.S.A.2002, 99, 6719−6724). TheSH3 domain is an all-β-struc-ture formed by the orthogo-nal sandwiching of a β-hair-pin (formed by the terminalsegments) and a three-

stranded antiparallel β-sheet.Using the program

CHARMM, the researchersmodeled the protein by con-sidering all of its heavy atomsand hydrogen atoms boundto nitrogen or oxygen. Con-tacts between residues wereconsidered to exist when sidechain-heavy atoms of twononadjacent residues werewithin 6 Å. Nonpolar residueswere predominantly selectedbecause mutations of theseresidues typically have thegreatest effect on overallfolding.

In agreement with exper-imental results, the simula-tion indicated that the pro-tein termini move throughseveral non-native positionsduring folding while the anti-parallel β-sheet is largelyfolded (see figure). The sim-ulation also indicated thatseveral residue side chainsform contacts with other sidechains that are not found inthe native structure. Thesenon-native interactions

occur predominantly in theturn regions and, in the caseof two loops, must return to

nativelike packing before fastfolding can occur.

Taken together, theseresults demonstrate that anatomic-resolution descrip-tion of the TSE can be devel-oped from MD simulations.

Directand SensitiveImmunodetectionTo perform immunodetec-tion of proteins separated ona PAGE gel, the proteins mustbe transferred to membranes(a Western blot), becausePAGE gels are impenetrableto antibodies. However, theefficiency of this transfer isvariable, leading to potential-ly significant disparitiesbetween the electrophoresisresults and the patterns andamounts of material that areimmunoprobed.

Agarose gels, by contrast,are permeable to antibodies.Thus, Gary Smejkal and hiscolleagues at Cleveland StateUniversity (Ohio) developed amethod for directly immuno-probing proteins following

agarose-gel electro-phoresis, without theneed for blots. Inaddition, they usedchemiluminescentimmunodetection asa sensitive alternativeto immunostaining(Electrophoresis 2002,23, 979−984).

Initially, theresearchers separatedfibrinogen and itsderivatives on stan-dard 1.5-mm-thickagarose gels, whichwere then incubatedwith horseradish per-oxidase (HRP)-conju-gated rabbit anti-

human fibrinogen antibod-ies. Subsequently, gels wereeither treated with luminol-based chemiluminescentsubstrates or stained with3,3′-diaminobenzidene(DAB). Although chemilumi-nescent labeling achieved adetection sensitivity of 500pg of fibrinogen per band,

DAB staining only detected5 ng/band. In addition, cer-tain fibrinogen oligomersand plasma fibrinogen multi-mers that were notdetectable with DAB wereeasily observed by chemilu-minescence.

Although the agarose gelmethod eliminates the risk ofmisrepresenting electrophor-etic separations due to blot-ting, other complicationsarise that generally do notoccur with Western blotanalysis. The greater thick-ness of the agarose gelsmakes complete washing ofunbound antibodies muchmore difficult than it is forblots, where immunoprobingis primarily limited to themembrane surface. Thisproblem resulted in notablebackground signals. In addi-tion, the probe time and theamount of antibody thatwere needed were signifi-cantly greater. Thus, using aspecialized frame, theresearchers compressed theagarose gel (derivatized with1.2% glyoxyl) down to athickness of 0.3 mm. Becausethis compression was per-formed prior to immuno-probing and after electro-phoresis, no backgroundchemiluminescence or stain-ing was observed and lesstime and material were nec-essary.

Using the compressedgels, the relative sensitivityof chemiluminescence re-mained at about 10 timesgreater than for staining;however, both approacheswere about 30 times less sen-sitive than the thick gel analy-sis. The researchers indicatedthat this was partly due to thelower HRP content of theantibody used in this part ofthe experiment. They pointedout, as well, that because nobackground noise was ob-served, the sensitivity couldbe improved by adding moreantibody and more chemilu-minescent substrates.

currents

JournalofProteomeResearch •Vol. 1, No. 4, 2002 301

Folding through the transition-state. Steps in one SH3 foldingpathway. (Adapted with permissionfrom Proc. Natl. Acad. Sci. U.S.A.2002,99, 6719−6724.Copyright 2002National Academy of SciencesU.S.A.)

currents

302 JournalofProteomeResearch •Vol. 1, No. 4, 2002

Rapid VirusIdentificationUsing M SVarious mass spectrometrictechniques have been usedin the past to characterizeboth known and unknownviruses. In most of thesemethods, proteolysis is per-formed on intact viruses forvarying incubation times,with protein molecularweights compared to those instandard databases. However,these techniques are bothtime-consuming and unreli-able for viruses that showfew biomarkers.

Catherine Fenselau andcolleagues at University ofMaryland (College Park) re-cently proposed a new bioin-formatics strategy to solvethis problem (Anal. Chem.2002, 74, 2529–2534). Theirsolution involves using adatabase of the calculatedmasses of peptides predictedfrom residue-specific pro-tease cleavage (e.g., bytrypsin) of all proteins ineach microorganism of inter-est. In this strategy, theexperimentally observedpeptide masses are searchedagainst the database to iden-tify the virus directly.

The researchers digestedSindbis virus AR339 (SIN)with a trypsin solution. Atvarious digestion times,samples were subjected toMALDI-MS analysis. Thethin-layer sample preparationtechnique was used forMALDI-TOF analysis, and theinstrument was internallycalibrated with known peakscorresponding to autolysistrypsin peptides.

After digestion, the scien-tists compared their resultswith a database they con-structed from the NationalCenter for BiotechnologyInformation proteome data-bases of proteins from six dif-ferent viruses: Lelystad virus,Sindbis virus (SIN) strainAR339, Ross River virus strainNB5092, human adenovirustype 5, tobacco mosaic virus

U2, and bacteriophage MS2.All experimentally observedpeptide masses from thedigested SIN solution werecompared with the masses ofthe in silico-generated trypticpeptides from each viruswithin a range of 1000−4000Da.

Digestion of the SIN virusfor only 2 min resulted in theobservation of more than 20tryptic peptides in a MALDIspectrum. Digestion of 5minutes or longer did notincrease the number ofobserved peptides.

Two algorithms weretested for identification: adirect score-ranking algo-rithmone that links thehighest number of matchedpeptide weights with the tar-get organismand an algo-rithm that evaluates theprobability of randommatching. The SIN virus was

unambiguously identified byeither approach.

The new method identi-fies whole virus samples inless than 5 minutes, using aMALDI-TOF MS with limitedresolution. The scientistsenvision the construction ofan on-line tryptic peptidedatabase for all viruses withknown genome sequences inthe future, incorporatingadditional information suchas protein copy number,post-translational modifica-

tions, and virus topology.

M icrofabricated CellSorterProteome complexity is oftendue to tissue heterogeneity,where each different cell typecontributes its own proteinmilieu. Thus, the isolation ofspecific cells from a mixturewould simplify proteomicanalysis. Traditionally, this hasbeen accomplished with tech-niques such as fluorescence-activated cell sorters (FACS),but these systems suffer fromthe need for large samples,high background fluores-cence, and the potential forcross-contamination.Withthis in mind, Stephen Quakeand colleagues at the Califor-nia Institute of Technology(Pasadena) developed an inte-grated microfabricated cellsorter using multilayer softlithography (Anal. Chem. 2002,74, 2451–2457).

The cell sorter has twolayers: a top piece that car-ries pneumatically controlledchannels for the pumps andvalves, and a bottom layerthat carries the microfluidiclines (which form a T-shape)for sample injection, collec-tion, and waste removal. Thesorter is placed on an invert-ed microscope, and a laser isused as the excitation source.A photomultiplier tubedetects the fluorescence ofpassing cells.

As the cells move alongthrough the sorter, they passthrough the fluorescencedetector at the junction of the

three sample lines. If no signalis detected, the pumps sendthe cell to the waste channel.If a cell fluoresces, however,the buffer flow is reversed,which sends the cell backthrough the detector. If fluo-rescence is detected a secondtime, the cell is then sent tothe collection channel.

The researchers testedtheir system on a mixed pop-ulation of E. coli expressingeither enhanced green fluo-rescent protein (EGFP) orp-nitrobenzyl (pNB) esterase.After sorting, the cells in thewaste and collection channelswere plated onto nutrientagar containing either ampi-cillin (upon which the EGFPcells would grow) or tetracy-cline (upon which the pNBcells would grow). Almost500,000 cells could be sortedin a single run, recovery yieldsreached 50% for some experi-ments, and cells wereenriched up to 89-fold.

The size of the integratedcell sorter means that verysmall samples can be handledquickly and with reducedbackground fluorescence ascompared to FACS. Further-more, the detection opticsoffer superior sensitivity, andthe simple fabrication andinexpensive materials makethe unit disposable, eliminat-ing problems of cross-contam-ination.

Celltrapping.A cell can be isolatedwithin the detection junction byreversing the buffer flow at eachdetection event. (Adapted fromAnal. Chem. 2002,74, 2451–2457.)

Waste Collection

Input

Cleave,separate,and search.Anew strategy for virus identificationinvolved partial proteolysis, massspectrometry, and database quer-ies. (Adapted from Anal. Chem.2002,74, 2529–2534.)