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Protein quantitation I: Overview(Week 8)1FractionationDigestionLC-MSLysisMS

Sample i Protein jPeptide kProteomic Bioinformatics Quantitation

2FractionationDigestionLC-MSLysisQuantitation Label-Free (Standard Curve)MS

Sample i Protein jPeptide k3FractionationDigestionLC-MSLysisQuantitation Label-Free (MS)MSMS

Assumption:

constant for all samples

Sample i Protein jPeptide k4HLQuantitation Metabolic Labeling

FractionationDigestionLC-MSLightHeavyLysisMSOda et al. PNAS 96 (1999) 6591Ong et al. MCP 1 (2002) 376

Assumption: All losses after mixing are identical for the heavy and light isotopes and

Sample i Protein jPeptide k5Comparison of metabolic labeling and label-free quantitation

G. Zhang et al., JPR 8 (2008) 1285-1292 Label free assumption:

constant for all samplesMetabolic labeling assumption:

constant for all samples and the behavior of heavy and light isotopes is identicalMetabolic

6G. Zhang et al., JPR 8 (2008) 1285-1292 Intensity variation between runsReplicates

1 IP1 Fractionation1 Digestion

vs

3 IP3 Fractionations1 Digestion

7How significant is a measured change in amount?It depends on the size of the random variation of the amount measurement that can be obtained by repeat measurement of identical samples.

8Protein ComplexesABACDDigestionMass spectrometry9

Tackett et al. JPR 2005Protein Complexes specific/non-specific binding

10Protein TurnoverKC=log(2)/tC, tC is the average time it takes for cells to go through the cell cycle, and KT=log(2)/tT, tT is the time it takes for half the proteins to turn over.

Move heavy labeled cells to light medium Heavy

LightNewly produced proteins will havelight label

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Super-SILAC

Geiger et al., Nature Methods 201012HLFractionationDigestionLC-MSLightHeavyLysisQuantitation Protein LabelingMSGygi et al. Nature Biotech 17 (1999) 994Assumption: All losses after mixing are identical for the heavy and light isotopes and

13HLFractionationDigestionLC-MSLysisMSLightRecombinantProteins (Heavy)Quantitation Labeled ProteinsAssumption: All losses after mixing are identical for the heavy and light isotopes and

14HLFractionationDigestionLC-MSLysisMSLightRecombinantChimericProteins (Heavy)Quantitation Labeled Chimeric ProteinsBeynon et al. Nature Methods 2 (2005) 587Anderson & Hunter MCP 5 (2006) 57315HLFractionationDigestionLC-MSLightHeavyLysisQuantitation Peptide LabelingMSGygi et al. Nature Biotech 17 (1999) 994Mirgorodskaya et al. RCMS 14 (2000) 1226

Assumption: All losses after mixing are identical for the heavy and light isotopes and

16HLFractionationDigestionLC-MSLightLysisSyntheticPeptides(Heavy)Quantitation Labeled Synthetic PeptidesMSGerber et al. PNAS 100 (2003) 6940

Enrichment withPeptide antibodyAssumption: All losses after mixing are identical for the heavy and light isotopes and

Anderson, N.L., et al. Proteomics 3 (2004) 235-4417FractionationDigestionLC-MSLysisMS/MSMSMSMS/MSQuantitation Label-Free (MS/MS)SRM/MRM18MS/MSSyntheticPeptides(Heavy)SyntheticPeptides(Heavy)LightHLMSHLMSMS/MSMS/MSMS/MSLLHHDigestionLC-MSLysis/FractionationQuantitation Labeled Synthetic Peptides19FractionationDigestionLC-MSLightHeavyLysisLHMSMS/MSQuantitation Isobaric Peptide LabelingRoss et al. MCP 3 (2004) 115420

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Isotope distributionsm/zm/zm/zIntensity22

Isotope distributionsPeptide massIntensity ratioPeptide massIntensity ratio23

Estimating peptide quantityPeak height

Curve fittingPeak areaPeak heightCurve fittingm/zIntensity24Time dimension

m/zIntensityTime

m/zTime25Sampling

Retention TimeIntensity26

5%Acquisition time = 0.05s5%Sampling27

Sampling28

Retention Time Alignment29Estimating peptide quantity by spectrum counting

m/zTimeLiu et al., Anal. Chem. 2004, 76, 4193

30What is the best way to estimate quantity?Peak height - resistant to interference- poor statistics

Peak area - better statistics - more sensitive to interferenceCurve fitting - better statistics- needs to know the peak shape- slow

Spectrum counting - resistant to interference- easy to implement- poor statistics for low-abundance proteins31

Examples - qTOF32

Examples - Orbitrap33

Examples - Orbitrap34AADDTWEPFASGK

IntensityIntensityIntensity

RatioRatio

012012Time35AADDTWEPFASGK

IntensityIntensityIntensity

m/zm/zm/z

GHI36YVLTQPPSVSVAPGQTAR

IntensityIntensityIntensity

RatioRatio012012Time37YVLTQPPSVSVAPGQTAR

IntensityIntensityIntensity

m/zm/zm/z38InterferenceAnalysis of low abundance proteins is sensitive to interference from other components of the sample.

MS1 interference: other components of the sample that overlap with the isotope distribution.

MS/MS interference: other components of the sample with same precursor and fragment masses as the transitions that are monitored. 39MS1 interference

40Data taken from CPTAC Verification Work Group Study 7.

10 peptides3 transitions per peptideConcentrations 1-500 fmol/l Human plasma background8 laboratories 4 repeat analysis per lab

Addona et al., Nature Biotechnol. 27 (2009) 633-641Quantitation using MRM

Addona et al., NBT 2009Peptide 1Peptide 241

Quantitation using MRM

Addona et al., NBT 2009Peptide 1Peptide 2Peptide 3Peptide 442Ratios of intensities of transitionsAddona et al., NBT 2009

Peptide 1Peptide 3Peptide 1Peptide 343Model: Noise and InterferenceIntensityCan the knowledge of the relative intensity of the transitions be used to correct for interference?

m/zNoise is a normally distributed increase or decrease in the intensity.

Interference is an increase in the intensity of one or more transitions.

44Detection of interference

Interference is detected by comparing the ratio of the intensity of pairs of transitions with the expected ratio and finding outliers.

Transition i has interference if

where Zthreshold is the interference detection threshold;

;

zji is the number of standard deviations that the ratio between the intensities of transitions j and i deviate from the noise;

Ii and Ij are the log intensities of transitions i and j;

rji is the median of the log intensity of transitions j and i; sji is the noise in the ratio.

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Error in quantitation after correction in presence of noise but no interferenceRelative noise = 0.2No interferenceRelative intensity of transitions: 1:1:1

46Corrections for interference

Relative ErrorCorrected Relative ErrorNo CorrectionPerfect Correction0047

Error in quantitation after correctionin presence of interference and noiseRelative noise = 0.2Interference in 1 out of 3 transitionsRelative intensity of transitions: 1:1:1No correctionCorrection (zth=2)48

Error in quantitation after correction in presence of interference and noiseRelative noise = 0.2Interference in 1 out of 3 transitionsRelative intensity of transitions: 1:1:1

Relative error before correction 0.3-0.7Relative error before correction 1.3-1.7

ztreshold = 0ztreshold = 1ztreshold = 2ztreshold = 349Error in quantitation after correction in presence of interference and noise

Interference in 2 out of 3 transitions

Interference in 1 out of 3 transitionszth = 2Relative noise = 0.2Relative intensity of transitions: 1:1:1

50Correction for MS2 interference

51Workflow for quantitation with LC-MS

StandardizationRetention time alignmentMass calibrationIntensity normalization

Quality ControlDetection of problems with samples and analysis

QuantitationPeak detectionBackground subtractionLimits for integration in time and massExclusion of interfering peaks52Takeaway MessageThere are many different ways to quantitate proteins choose the one that is appropriate for your application.

In general the earlier you can introduce isotopic labels the better the accuracy.

Always monitor for interference.53Protein quantitation I: Overview(Week 8)54