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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