Protein quantitation I: Overview (Week 6) Proteomic Bioinformatics - PowerPoint PPT Presentation

Protein quantitation I: Overview (Week 6)

Proteomic Bioinformatics – Quantitation Sample i C ij Protein j Lysis p L Peptide k p ij Fractionation Pr p MS ij p ik D Digestion ijk p Pep MS α k I ik LC-MS ik p LC ik   ∑ α L Pr D Pep LC MS = p p p p p p I C     ik k ij ij ij ijk ik ik ik j I = k ik C α L Pr D Pep LC MS ij p p p p p p k ij ij ijk ik ik ik

Quantitation – Label-Free (Standard Curve) Sample i Protein j Peptide k Lysis Fractionation Digestion LC-MS k = ≈ β C I I f ( ) ik ik ij MS

Quantitation – Label-Free (MS) Sample i Protein j Peptide k Lysis Assumption: α L Pr D Pep LC MS p p p p p p Fractionation k ij ij ijk ik ik ik constant for all samples Digestion = C / C I / I i i i i j j j j LC-MS n m n m MS MS

Quantitation – Metabolic Labeling L H C C Light Heavy i n j i m j M M p p Lysis i m j i n j Assumption: All Fractionation losses after mixing are identical for the Digestion Sample i heavy and light Protein j isotopes and M M LC-MS ≈ Peptide k p p i i j j n m MS H L H I L I i n i m k k Oda et al. PNAS 96 (1999) 6591 Ong et al. MCP 1 (2002) 376

Comparison of metabolic labeling and label-free quantitation Label free assumption: SILAC Metabolic α L Pr D Pep LC MS Label-Free p p p p p p k ij ij ijk ik ik ik constant for all samples Metabolic labeling assumption: M p ij constant for all samples and -1 -0.5 0 0.5 1 log2(ratio) the behavior of heavy and light isotopes is identical G. Zhang et al., JPR 8 (2008) 1285-1292

Intensity variation between runs Replicates 1 IP 1-1-1 1 Fractionation 3-3-1 1 Digestion vs 3 IP 3 Fractionations 1 Digestion -1 -0.5 0 0.5 1 log2(ratio) G. Zhang et al., JPR 8 (2008) 1285-1292

How significant is a measured change in amount? It depends on the size of SILAC the random variation of Label-Free the amount measurement that can be obtained by repeat measurement of identical samples. -1 -0.5 0 0.5 1 log2(ratio)

Protein Complexes A D A C B Digestion Mass spectrometry

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

Protein Turnover Heavy Light Move heavy Newly produced labeled cells to proteins will have light medium light label dC H ( t ) K K C H j ( ) ( t ) = − + dt C T j K K e C C H H ( ) t − + ( t ) ( ) ⇒ = 0 C T C C C L H H j j ( t ) ( t ) ( ) + = 0 j j j K C =log(2)/t C , t C is the average time it takes for cells to go through the cell cycle, and K T =log(2)/t T , t T is the time it takes for half the proteins to turn over. I I H L ( t ) ( t ) + 1 1 j j log( ) t ( ) log( ) = + 2 t t I H ( t ) C T j

Super-SILAC Geiger et al., Nature Methods 2010

Quantitation – Protein Labeling Assumption: All Lysis losses after mixing Light Heavy are identical for the Fractionation heavy and light isotopes and p p p p L M L M ≈ Digestion i i i i j j j j n n m m LC-MS MS H L Gygi et al. Nature Biotech 17 (1999) 994

Quantitation – Labeled Proteins Recombinant Proteins (Heavy) Lysis Assumption: All Light losses after mixing Fractionation are identical for the heavy and light Digestion isotopes and p p p L M M ≈ i i i j j j n n m LC-MS MS H L

Quantitation – Labeled Chimeric Proteins Recombinant Chimeric Proteins (Heavy) Lysis Fractionation Light Digestion LC-MS MS H L Beynon et al. Nature Methods 2 (2005) 587 Anderson & Hunter MCP 5 (2006) 573

Quantitation – Peptide Labeling Assumption: All losses after mixing are identical for the heavy and light Lysis isotopes and Fractionation L Pr D M ≈ p p p p i i i i j j jk k n n n n Digestion L Pr D M ≈ p p p p Light Heavy i i i i j j jk k m m m m LC-MS MS H L Gygi et al. Nature Biotech 17 (1999) 994 Mirgorodskaya et al. RCMS 14 (2000) 1226

Quantitation – Labeled Synthetic Peptides Assumption: All losses after mixing are identical for the heavy and light Lysis isotopes and L Pr D M M ≈ p p p p p Fractionation i i i i j j jk k sk n n n n Synthetic Digestion Peptides Light Enrichment with (Heavy) Peptide antibody LC-MS Anderson, N.L., et al. Proteomics 3 (2004) 235-44 MS H L Gerber et al. PNAS 100 (2003) 6940

Quantitation – Label-Free (MS/MS) Lysis Fractionation Digestion LC-MS SRM/MRM MS/MS MS MS MS/MS

Quantitation – Labeled Synthetic Peptides Light Lysis/Fractionation Synthetic Synthetic Digestion Peptides Peptides (Heavy) (Heavy) LC-MS MS MS H H L L MS/MS L MS/MS H MS/MS L MS/MS H

Quantitation – Isobaric Peptide Labeling Lysis Fractionation Digestion Light Heavy LC-MS MS MS/MS H Ross et al. MCP 3 (2004) 1154 L

Quantitation – Label-Free (MS) Quantitation – Label-Free (MS/MS) Quantitation – Label-Free (Standard Curve) Lysis Lysis Lysis Fractionation Fractionation Fractionation Digestion Digestion Digestion LC-MS LC-MS LC-MS MS MS MS/MS MS MS MS/MS MS Quantitation – Metabolic Labeling Quantitation – Protein Labeling Quantitation – Labeled Chimeric Proteins Recombinant Light Chimeric Heavy Proteins (Heavy) Lysis Lysis Lysis Light Heavy Fractionation Fractionation Fractionation Light Digestion Digestion Digestion LC-MS LC-MS LC-MS MS MS MS H H H L L L Quantitation – Peptide Labeling Quantitation – Isobaric Peptide Labeling Quantitation – Labeled Synthetic Peptides Lysis Lysis Lysis Fractionation Fractionation Fractionation Synthetic Digestion Digestion Digestion Light Light Light Peptides Heavy Heavy (Heavy) LC-MS LC-MS LC-MS MS MS MS/MS MS H H H L L L

Isotope distributions m = 1035 Da m= 1878 Da m = 2234 Da Intensity m/z m/z m/z

Isotope distributions Intensity ratio Intensity ratio Peptide mass Peptide mass

Estimating peptide quantity Peak height Peak height Curve fitting Curve fitting Intensity Peak area m/z

Time dimension Intensity m/z Time Time m/z

Sampling Intensity Retention Time

Sampling 140 3 points 120 100 80 60 5% 40 20 0 0.8 0.85 0.9 0.95 1 30 3 points 25 20 5% 15 10 5 0 0.8 0.85 0.9 0.95 1 Acquisition time = 0.05 σ

Sampling 1.1 1 Thresholds (90%) 0.9 0.8 0.7 0.6 0.5 1 2 3 4 5 6 7 8 9 10 # of points

Retention Time Alignment

Estimating peptide quantity by spectrum counting Time m/z Liu et al., Anal. Chem. 2004, 76, 4193

What is the best way to estimate quantity? Peak height - resistant to interference - poor statistics Peak area - better statistics - more sensitive to interference Curve fitting - better statistics - needs to know the peak shape - slow Spectrum counting - resistant to interference - easy to implement - poor statistics for low-abundance proteins

Examples - qTOF

Examples - Orbitrap

Examples - Orbitrap

Intensity AADDTWEPFASGK Intensity Intensity 2 2 Ratio 1 1 0 0 2 2 Ratio 1 1 0 0 Time

Intensity AADDTWEPFASGK Intensity Intensity G m/z H m/z I m/z

Intensity YVLTQPPSVSVAPGQTAR Intensity Intensity 2 2 Ratio 1 1 0 0 2 2 Ratio 1 1 0 0 Time

Intensity YVLTQPPSVSVAPGQTAR Intensity Intensity m/z m/z m/z

Interference Analysis 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.

MS1 interference

Quantitation using MRM 1000 Peptide 1 Measured concentration [fmol/ul] 100 Data taken from CPTAC Verification Work Group Study 7. 10 10 peptides line 1 tr1 3 transitions per peptide tr2 tr3 Concentrations 1-500 fmol/ μ l 0.1 Human plasma background 1 10 100 1000 Actual concentration [fmol/ul] 8 laboratories 1000 4 repeat analysis per lab Peptide 2 Measured concentration [fmol/ul] Addona et al., Nature 100 Biotechnol. 27 (2009) 633-641 10 line 1 tr1 tr2 tr3 0.1 1 10 100 1000 Actual concentration [fmol/ul] Addona et al., NBT 2009

Quantitation using MRM 1000 1000 Peptide 1 Peptide 3 Measured concentration [fmol/ul] Measured concentration [fmol/ul] 100 100 10 10 line line 1 1 tr1 tr1 tr2 tr2 tr3 tr3 0.1 0.1 1 10 100 1000 1 10 100 1000 Actual concentration [fmol/ul] Actual concentration [fmol/ul] 1000 1000 Peptide 2 Peptide 4 Measured concentration [fmol/ul] Measured concentration [fmol/ul] 100 100 10 10 line line 1 tr1 tr1 tr2 tr2 tr3 tr3 0.1 1 1 10 100 1000 1 10 100 1000 Actual concentration [fmol/ul] Actual concentration [fmol/ul] Addona et al., NBT 2009