Pinpointing phosphorylation sites using Selected Reaction Monitoring and Skyline

Transcription

1 Pinpointing phosphorylation sites using Selected Reaction Monitoring and Skyline Christina Ludwig group of Ruedi Aebersold, ETH Zürich

2 The challenge of phosphosite assignment Peptides Phosphopeptides MS/MS spectra S T S R A G A MS/MS spectra S T* S A G A R AS*GS*T*AR phosphopeptide identification ASGSTAR peptide identification ASGST*AR phosphosite assignment 1

3 The case study: Phosphorylation changes upon shock before shock after shock 204 metabolic yeast enzymes yeast cell cultures steadystate protein extraction digestion (trypsin) phosphoenrichment (TiO 2 ) 2

4 Pinpointing Determination The case study: of phosphorylation Phosphorylation stoichiometries sites changes using SRM upon shock phosphorylation results table phosphopeptide sequence before shock after shock ratio YGGHS[p]MSDPGTTYR KGS[p]MADVPK shotgun LCMS/MS (LTQOrbi) labelfree quantification SASAVS[p]LPAK

5 Ambiguities and missing values in discoverydriven data sets Gpd2 = Glycerol3phosphate dehydrogenase 2 Peptide sequence Peptide sequence phosphosite phosphosite PepP probability PepP probability before shock A before shock A before shock B before shock B before shock C before shock C after shock A after shock A after shock B after shock B after shock C S[p]DSAVSIVHLK [S70] #DIV/0! SDS[p]AVSIVHLK [S72] E03 SDSAVS[p]IVHLK [S75] E01 S[p]DSAVS[p]IVHLK [S70],[S75] #DIV/0! SDS[p]AVS[p]IVHLK [S72],[S75] E02 Gpd1 = Glycerol3phosphate dehydrogenase 1 after shock C SS[p]SSVSLK [S23] #DIV/0! SSS[p]SVSLK [S24] E03 SSSS[p]VSLK [S25] E03 SSSSVS[p]LK [S27] E01 SSS[p]SVS[p]LK [S24],[S27] E02 ratio ratio Ttest Ttest Are all identified phosphosites truly occurring in the sample? Do the obtained quantitative results reflect true biological changes? Targeted phosphoproteomics using SRM and Skyline 4

6 (Phospho)SRM workflow carried out in Skyline (phospho) peptide selection transition selection SRM measurement crude synthetic heavy phosphopeptides (JPT technologies) Gpd1 peptide sequence S[p]SSSVSLK [S22] Gpd2 peptide sequence S[p]DSAVSIVHLK phosphosite phosphosite [S70] SS[p]SSVSLK [S23] SDS[p]AVSIVHLK [S72] SSS[p]SVSLK [S24] SDSAVS[p]IVHLK [S75] SSSS[p]VSLK [S25] SSSSVS[p]LK [S27] 5

7 Getting phosphopeptides into Skyline (phospho) peptide selection 6

8 Getting phosphopeptides into Skyline (phospho) peptide selection All Uniprot modifications available 7

9 (Phospho)SRM workflow carried out in Skyline (phospho) peptide selection transition selection SRM measurement automatic selection of the yion and bion series (> 300 Da) using the transition filter settings in Skyline shared transitions unique transitions peptide sequence phosphosite precursor m/z y10 b1 y9 b2 y8 b3 y7 b4 y6 b5 y5 b6 y4 b7 y3 b8 y2 b9 y1 b10 S[p]DSAVSIVHLK [S70] SDS[p]AVSIVHLK [S72] SDSAVS[p]IVHLK [S75]

10 (Phospho)SRM workflow carried out in Skyline (phospho) peptide selection transition selection SRM measurement of synthetic peptides 9

11 Targeting of synthetic phosphopeptides for Gpd2 SRM measurement individual measurements of synthetic peptides [S70] [S72] [S75] peptide mixture [S70], 14.6 min [S72], 14.3 min [S75], 14.9 min all phosphopeptide forms are separable in retention time 10

12 Targeting of synthetic phosphopeptides for Gpd1 SRM measurement [S22] [S23] [S24] [S25] [S27] individual measurements of synthetic phosphopeptides peptide mixture [S24],[S25], 5.6 min [S23], 5.8 min [S22], 6.0 min [S27], 6.3 min phosphopeptide forms [S24] and [S25] are not separable in retention time. Other peptide forms are strongly overlapping but distinguishable. 11

13 Discrimination of [S24] [S25] despite coelution SRM measurement peptide sequence phosphosite precursor m/z y7 y6 y5 y4 y3 rt [min] S[p]SSSVSLK [S22] SS[p]SSVSLK [S23] SSS[p]SVSLK [S24] SSSS[p]VSLK [S25] SSSSVS[p]LK [S27] [S22] [S23] [S24] [S25] [S27] y [S24] [S23] [S22] 12

14 Discrimination of [S24] [S25] despite coelution SRM measurement peptide sequence phosphosite precursor m/z y7 y6 y5 y4 y3 rt [min] S[p]SSSVSLK [S22] SS[p]SSVSLK [S23] SSS[p]SVSLK [S24] SSSS[p]VSLK [S25] SSSSVS[p]LK [S27] [S22] [S23] [S24] [S25] [S27] y [S25] [S27] 13

15 intensity Retention time information is important for pinpointing phosphosites Phosphopeptide: XXX[S4]X[S6]X[S8]XX [S4] [S6] [S8] retention time retention time retention time [S4] separable in retention time [S6] [S8] coeluting [S4] [S6] [S8] X Y Z transitions usable for identification and quantification X X X [S4] [S6] [S8] [S4] [S6] [S8] not specifically identifiable and quantifiable X Y Z X X 14

16 accurate RT The irt concept implemented into Skyline The problem: The solution: Accurately measured empirical retention times are dependent on the setup of the currently used chromatographic system Usage of a set of calibration peptides to normalize all peptide retention times to a dimensionless irt value 14.8 min calibration peptides 14.5 min 15.1 min irt Consequence in practice: retention times need to be determined over and over again Consequence in practice: once an irt value is determined, the respective peptide can be scheduled and identified on any LC system Escher C. et al., Proteomics, 2012, 12, Monday AM, Poster 624, Escher C. et al. 15

17 Quantification of endogenous phosphopeptides Gpd2 phosphoenriched yeast total cell extract synthetic, isotopicallylabeled phosphopeptides endogenous (light) synthetic (heavy) [S72] [S75] [S70] light heavy light heavy [S72] [S75] 16

18 ratio light/heavy ratio light/heavy Quantification of endogenous phosphopeptides Gpd2 phosphoenriched yeast total cell extract synthetic, isotopicallylabeled phosphopeptides endogenous (light) synthetic (heavy) [S72] [S75] [S70] [S72] [S75] before 30 min after shock 1.5fold downregulation (ttest pvalue = 0.01) before 30 min after shock 1.7fold downregulation (ttest pvalue = 0.01) 17

19 Quantification of endogenous phosphopeptides Gpd1 phosphoenriched yeast total cell extract synthetic, isotopicallylabeled phosphopeptides endogenous (light) synthetic (heavy) [S24] [S27] [S24] [S25] light heavy light heavy [S23] [S22] [S27] 18

20 ratio light/heavy ratio light/heavy Quantification of endogenous phosphopeptides Gpd1 phosphoenriched yeast total cell extract synthetic, isotopicallylabeled phosphopeptides endogenous (light) synthetic (heavy) [S24] [S25] [S24] [S27] [S23] [S22] [S27] before shock 30 min after before 30 min after shock 7.2fold upregulation (ttest pvalue = 2.1E4) 3.2fold upregulation (ttest pvalue = 2.4E4) 19

21 ratio light/heavy Quantification of a doubly phosphorylated peptide Gpd1 Spikein experiment (heavy crude phosphopeptides) SSS[p]SVS[p]LK [S24,27] endogenous (light) synthetic (heavy) light [S24,S27] heavy [S24,S27] before shock after 3.5fold upregulation (ttest pvalue = 4.4E4) 20

22 Takehome messages Targeted analysis of phosphorylation using SRM provides quantitative data of high quality, accuracy and reproducibility Requirement: A priori knowledge Specific phosphorylationsites can be pinpointed with high confidence Requirement: Learn the chromatographic behavior of phosphopeptide forms from synthetic reference peptides Application of the irt concept improves acquisition scheduling and peptide identification Requirement: Consistent use of retention time calibration peptides 21

23 Thank you Collaborators Ana Paula Oliveira Paola Picotti TSQsupport Mariette Matondo Nathalie Selevsek Ruedi Aebersold and the whole Aebersold lab Brendan MacLean Alana Killeen Lukas Reiter Oliver Rinner

24 Further complications for a phosphoanalysis with SRM: Neutral Loss H 3 PO 4 1. Loss of H 3 PO 4 = 98 Da example: y7 O y7 S D S A V S I V H L K S D S A V S I V H L K phosphoserine CID MS/MS H 3 PO 4 serine CID MS/MS H 2 O y7 backbone fragmentation & H 3 PO 4 loss y7 backbone fragmentation & H 2 O loss on S75 V S I V H L K transition coordinates V S I V H L K dehydroalanine dehydroalanine Q Q Conclusion For peptides comprising several possibly phosphorylated residues phosphosite assignments based exclusively on H 3 PO 4 neutral loss fragment ions can be erroneous, because their occurrence can also be due to a waterloss of a nonphosphorylated serine residue.

25 example for H 3 PO 4 (98 Da) phosphate loss interference peptide sequence Phosphosite precursor m/z y10 y10h 3 PO 4 b1 b1h 3 PO 4 y9 y9h 3 PO 4 b2 b2h 3 PO 4 y8 y8h 3 PO 4 b3 b3 H 3 PO 4 y7 y7h 3 PO 4 b4 b4h 3 PO 4 y6 y6h 3 PO 4 b5 b5h 3 PO 4 y5 y5h 3 PO 4 b6 b6h 3 PO 4 y4 y4h 3 PO 4 b7 b7h 3 PO 4 y3 y3h 3 PO 4 b8 b8h 3 PO 4 y2 y2h 3 PO 4 b9 b9h 3 PO 4 y1 y1h 3 PO 4 b10 b10h 3 PO 4 rt[mi n] S[p]DSAVSIVHLK [S70] SDS[p]AVSIVHLK [S72] SDSAVS[p]IVHLK [S75] [S70] [S72] [S75]

26 Further complications for a phosphoanalysis with SRM: Neutral Loss HPO 3 2. Loss of HPO 3 = 80 Da example: O y7 y7 phosphoserine CID MS/MS HPO 3 S D S A V S I V H L K V S I V H L K y7 backbone fragmentation S D S A V S I V H L K V S I V H L K y7 backbone fragmentation & HPO 3 loss on S75 transition coordinates serine Q Q Conclusion For peptides comprising several possibly phosphorylated residues phosphosite assignments based exclusively on fragment ions NOT carrying the phosphate group can be erroneous, because their occurrence can also be due to a neutral loss of HPO 3. 17

27 The irt concept implemented into Skyline 17

28 The irt concept implemented into Skyline 18

29 The irt concept implemented into Skyline 19

30 The irt concept implemented into Skyline 20