Development of a multiplexed quantitative proteomics assay ... · Bins (% CV) A) B) 0.00 0.50 1.00...
Transcript of Development of a multiplexed quantitative proteomics assay ... · Bins (% CV) A) B) 0.00 0.50 1.00...
Overview
Introduction
Method
Results
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median CV = 16.6% median CV = 26.0%
median CV = 20.8% median CV = 25.2%
AlbuminCystain-Cβ-2-microglobulinClusterinLipocalin-2VEGFAUromodulin
13%
21%
6%47%
3%10%
Fribinogen LDHCK-BCK-MFABP3AST1
PARVALBUMIN-α
Serum Amyloid A VCAM-1
HaptoglobinThrombospondin-1
CRP
FDA, EMEA
PROTEINS WITH KNOWNOR SUSPECTED ORGAN TOXICITY ASSOCIATION
LITTERATURE SEARCHDISCOVERY STUDIES
BIOMARKER TARGET SELECTIONGenerate a comprehensive list of
MSAIQAAWPSGTECIAKYNFHGTAEQDLPFCKGDVLTIVAVTKDPNWYKAKNKVGREGIIPANYVQKREGVKAGTKLSLMPWFHGKITREQAERLLYPPETGLFLVRESTNYPGDYTLCVSCDGKVEHYRIMYHASKLSIDEEVYFENLKMQLVEHYTSDADGLCTRLIKPKVMEGTVAAQDEFYRSGWALNMKELKLLQTIGKGEFGDVMLGDYRGNKVAVKCIKNDATA…
Proteotypic:no Met or Cys residues, m/z within scanning range of instrument.
MULTIPLEXED MRM ASSAY OPTIMIZATION
QUALIFIEDBIOMARKERS OF
NEPHROTOXICITY
mixture to a reverse-phase (C18) HPLC system coupled online to a triple quadrupole instrument. .
Q1:Stage one mass filtertargets the precursor
Q2:
isolated and fragmentedin collision cell
Q3:Stage two mass
filter targets the sequence specific fragment ion
ASSAY PROCEDURE
URINE - two formatTube-based format for: n ≤ 30Plate-based format for: n > 30
Plate-based format for:20-40 samples/plate
PLASMA/SERUM
SAFETY BIOMARKERS IN PLASMA/SERUM: PLASMA/SERUM-BASED MRM ASSAY REPRODUCIBILITY
Round 1 Round 2
Round 3 Overall
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DETECTED SAFETY BIOMARKERS IN RAT PLASMA/SERUM SAMPLES
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Kidney Heart Liver Skeletal muscle Vascular system Brain
MyoglobinLDHCK-BCK-MFABP3
AST1MDH-1
PNPPON-1
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3.0E+07
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1. 0.2u filtration of samples to remove particulates2. Depletion of the 7 most abundant proteins using rat IgY-7 immuno-affinity resin.3. Enzymatic digestion of proteins4. Desalting5. MRM analysis
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median CV = 6.1%
SAFETY BIOMARKERS IN URINE: URINE-BASED MRM ASSAY REPRODUCIBILITY
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median CV = 27.1%
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B) Protein precipitation (tube-based format)n=20
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median CV = 27.9%
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DETECTED KIDNEY SAFETY BIOMARKERS IN RAT URINE SAMPLES
SAFETY BIOMARKERS IN URINE (CONT’D): PROOF-OF-CONCEPT: EFFECT OF A NEPHROTOXIC AGENT ON RENAL BIOMARKERS
Protein precipitation (plate-based format)n=72
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ALBUMIN
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UROMODULIN
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FLAVOPROTEIN
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EINE
LIGASE α-ENOLA
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78 KDA GLUCOSE
REGULATED PROTEIN
ARGININ
OSUCCIN
ATE
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BIFUNCTIO
NAL
PROTEIN NCOAT
HOMOGENTISATE
1, 2-D
IOXYGENASE AMBP
HISTONE H2A
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PROTEIN 1
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Conclusions
Sample #
Rat urine samples were precipitated using the plate-based format and analyzed by MRM. Parameters and instrument setup were optimized for maximum sensitivity. In total, 29 uri-nary proteins were detected, including 5 qualified biomarkers of nephrotoxicity, 6 bio-markers currently under investigation for qualification by the PSTC. The remaining 18 de-tected proteins are classified as exploratory biomarkers and can be used to add value to the characterization of the nephrotoxic insult. .
A protein precipitation procedure was developed and optimized for the analysis of urine samples by MS. The median coefficient of variation was calculated for each detected tran-sition using A) (left panel) a plate-based precipitation format and B) (left panel) a tube-based precipitation format and represents a measure of the variation caused by the over-all process. The data shows that the protein precipitation procedure is reproducible using both formats. Figure C (left panel) shows that the protein precipitation procedure is also comparable, in terms of reproducibility, to ultrafiltration. Figures A, B, C (right panel) show the median coefficient of variation calculated for 5 internal standard peptides spiked in each sample and constitutes a measure of the technical variation from the instrument during analysis. .
Development of a multiplexed quantitative proteomics assay for the predictive assessment of drug-induced organ toxicity in the pre-clincal settingMarlene Gharib 1,2, Pierre Thibault 2, Michael Schirm 2, Andrew Nelson 3, Daniel Chelsky 2, Lorella Di Donato 2, Laura McIntosh 2
1 Université de Montréal, Montrél, Québec, Canada2 Caprion Proteome Inc., Montréal, Québec, Canada3 ITR Canada, Baie d’Urfée, Québec, Canada
Rat urine from control samples (10 technical replicates of pooled normal rat urine) and kidney disease samples (baseline, vehicle, low dose and high dose treatment) were pre-pared using the plate-based protein precipitation procedure and analyzed by MRM. The median coefficient of variation calculated (Figure A) for all detected transitions in the tech-nical replicates show that the overall processe for all samples showed good reproducibil-ity. Figure B shows the median coefficient of variation calculated for 5 internal standard peptides spiked in each sample and constitutes a measure of the technical variation from the instrument during analysis of all study samples. The effect of the nephrotoxic agent on qualified kidney biomarkers (Figure C), on kidney biomarkers currently under inverstiga-tion for qualificatioon (Figure D) and exploratory biomarkers (Figure E) are shown. Trends observed for albumin, kim-1, b-2-microglobulin, clusterin, cystatin-C, Ostopontin, a-GST and lipocalin by MRM are in agreement with trends observed using an antibody-based method and corresponds to those reported in the literature. .
The established MS technology of multiple reaction monitoring (MRM) can be used in a multiplexed format to specifically target and reproducibly quantify hundreds of can-didate protein biomarkers in a single sample. In the present study, the development of a multiplexed MRM safety biomarker panel assay for the rapid profiling of drug-induced organ toxicity in rat plasma/serum and urine samples is described. .
Drug-induced organ toxicity is the primary reason for the withdrawal of drugs from the market and the failure of lead compounds during drug development. Detection of organ toxicity early in the development pipeline would not only reduce the cost of drug develop-ment but also prevent toxicity to patients. Limitations of traditional approaches highlight the need for more sensitive and specific tools to predict drug toxicty. With the recent regu-latory qualification of seven renal safety biomarkers for use during drug development, bio-markers are increasingly viewed as potential means for providing accurate and sensitive toxicity assessments. Here we have employed an antibody-free, highly multiplexed targeted mass spectrometry approach, known as multiple reaction monitoring (MRM), to quantitatively measure changes in organ-specific safety biomarkers. The predictive value of our biomarker panel was tested on urine samples from rats that were treated with a known nephrotoxic agent. The present MRM assay has the potential to become standard practice for pre-clinical drug safety assessments and shows great promise in improving the development of safer drugs. .
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The following chart illustrates all safety biomarkers detected in plasma and/or serum. In total, 78 safety biomarkers covering all organs included in our panel were detected. Selected examples for each organ are presented. .
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An antibody-free highly multiplexed MRM assay was succesfully developed to measure important biomarkers of hepatotoxicity, nephrotoxicity, cardiotoxicity as well as muscu-lar, vascular and neurotoxicity in rats. The overall assay is very reproducible and sensi-tive allowing the detection of major biomarkers currently under investigation as well as all qualified kidney kidney injury biomarkers in serum and urine, respectively. The assay requires a minimal amount of sample (3 ul for plasma/serum and 100 ul for urine) and involves a simple plate-based depletion or protein precipitation procedure, which is ad-vantageous in terms of cost and thoughput. The MRM assay was first evaluated using urine samples from rats that were treated with a known nephrotoxic agent. Expression analysis has demonstrated that kidney injury biomarkers were modulated following tox-icity and are in agreement with literature. Caprion is ready to offer screening services for the panel of nephrotoxic safety biomarkers for relative quantification in urine and is presently developing abslolute quantitation using heavy atom-labeled petides. Finally, application of our panel of safety biomarkers for use in clinical drug development studies is also underway. .
median CV = 22.7%
median CV = 8.1%
median CV = 10.4%
median CV = 12.1%
The IgY-7 plate-based immuno-affinity depletion protocol was optimized for maxi-mum reproducibility. Figure A shows the median coefficient of variation calculated for each detected transition for each depletion round and represents a measure of the variation caused by the overall assay procedure. Figure B shows the median coeffi-cient of variation calculated for 5 internal standard peptides spiked in each depleted sample and constitutes a measure of the technical variation caused by the instrument only. The data shows that the plasma/serum based assay is reproducible as the calcu-lated CV values are low. .
937 peptides for optimization in
one multiplexed run
1. Protein precipitation2. Enzymatic digestion of proteins3. Desalting6. MRM analysis
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# 945
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0.0E+005.0E+051.0E+061.5E+062.0E+062.5E+063.0E+063.5E+064.0E+064.5E+065.0E+06
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median CV = 16.0%
Bins (% CV)
Sample #
Sample #
Sample #
Albuminβ-2-microglobulin
Cystatin-CClusterin
KIM-1
RBP4Lipocalin-2 (NGAL)
Osteopontinπ-GSTα-GST
Calbindin
UromodulinAquaporin-1
EGFα-2-HS-glycoproteinMajor urinary protein
CTGFFABPH
λ-crystallinElectron transfer flavoprotein α
GAPDHGlutamate-cysteine ligase
α-enolase78 KDa glucose regulated protein
Argininosuccinate synthaseBifunctional protein NCOAT
PSTC QUALIFICATION ONGOING
GLOBAL MARKERS OF KIDNEY INJURY(EXPLORATORY)
Selenium-binding proteinHomogentisate 1,2-dioxygenase
AMBPHISTONE H2A
QUALIFIED BIOMARKERS OF NEPHROTOXICITY (PSTC, FDA, EMA, PDMA)