Protein Phosphorylation

Transcription

1 Phosphorylation Site Mapping 1

2 Protein Phosphorylation Reversible protein phosphorylation is the most common signal transduction event. Kinases catalyze the transfer of a γ-phosphate group from ATP to a Ser, Thr, or Tyr residue in a substrate protein. Phosphatases catalyze the removal of a phosphate group from ps, pt, or py in a substrate protein. As many as 1/3 of proteins thought ht to be phosphorylated. h An estimated 2-5% of all proteins thought to be kinases. Relative abundance of phosphoresidues: pser: 90% pthr: 10% ptyr: 0.05% 05% 2

3 Phosphorylation Analysis: The Relevant Questions How heavily phosphorylated is the protein? (stoichiometry) On which residues is the protein phosphorylated? (site identification) How heavily is each site phosphorylated? (sitespecific stoichiometry) Phosphoproteomics: How does the phosphoproteome change in response to a stimulus? Challenges low levels of phosphorylated proteins low phosphorylation stoichiometry transient modification 3

4 Phosphorylation Analysis: The Toolbox Affinity Purification Methods Phosphoantibody, IMAC, TiO 2, SAX ESI/MALDI-MSMS Tandem MS CID, ETD, marker ions product/precursor ion scanning Chemical Labeling Methods Isolation, relative quantitation 4

5 Phosphorylation Stoichiometry from an Intact Protein Molecular Weight (ESI) CFM-S T90 Hassell, A q3t (8.482) Sm (Mn, 3x5.00); Cm (437:451) Recombinant Kinase Autophosphorylation CFM-S T90 Hassell, A TOF MS ES+ q3t (8.482) M1 [Ev-65849,It9] (Gs,0.750,1378:1898,2.00,L40,R20); Sm (Mn, 3x5.00); % % Moles Phosphate/Mole Protein 80 Da Spacing/Phos 0 m/z 0 mass Need purified protein; more of a challenge by MALDI-Tof to resolve the 80Da difference 5

6 Mapping of Phosphorylation Sites Sample protein or proteome Digest IMAC, TiO 2 SAX, etc. Marker Ions, Neutral Losses, Peptide Sequence Isolate ppeptides MS Analysis Identify ppeptides/ Assign Site MALDI, LC/MS, LC/MS/MS MS Analysis Identify ppeptides/ Assign Site Marker Ions, Neutral Losses, Peptide Sequence Workflow taken depends upon tools, sample, coverage requirements 6

7 Mapping of Phosphorylation Sites Phosphoprotein is cleaved (digested) with a protease. Use of multiple proteases of differing specificity can increase coverage of the protein sequence. Cleavage with Trypsin H 2 N Y R K K Y R P Y K K P Y K COOH Cleavage with Asp-N H 2 N Y D Y D P Y D D P Y D COOH +80 Da mass shift per phosphate group added 7

8 Metastable Decomposition of ps and pt in MALDI-MS Abundant Ions Observed Include: [MH-H 3 PO 4 ] + (or MH-98) and [MH-HPOHPO + 3 ] (or MH-80) Matrix effects: αhca vs DHB Annan et al., Anal. Chem., 1996, 68,

9 Metastable Decomposition of py in MALDI-MS For ptyr containing peptides: [MH-H 3 PO 4 ] + <<< than for pser or pthr (MH-98) [MH-HPO 3 ] + < than for pser or pthr (MH-80) Annan et al., Anal. Chem., 1996, 68, Can also analyze digests/ fractions before and after treatment with Calf Intestinal Phosphatase (-80Da shift with removal of phosphate 9 group)

10 Phosphorylation Site Mapping by Data Directed Acquisition iti (DDA) Database Searching shotgun approach Data directed acquisition (automated LC/MS/MS) of product ion spectra of all peptides total digest or after phosphopeptide enrichment Search (Mascot, Sequest) all spectra against protein database allowing for Ser, Thr, or Tyr phosphorylation Database search tentatively ID s phosphopeptides and assigns sites if possible User confirms phosphorylation assignment Challenges Complex proteomes Limitations of DDA Poor quality CID spectra for some ps/pt peptides 10

11 Phosphorylation Site Mapping by DDA/Database Searching- Abl Phosphorylation of PLCγ Sample: GST-PLCγ/Abl kinase reaction mix Analysis by data directed LC/MS/MS Product ion spectra searched with variable modification for phosphorylation 4 Phosphorylation sites tentatively ID d 1. P10686 Mass: Total score: 873 Peptides matched: 52 PIP4_RAT 1-PHOSPHATIDYLINOSITOL-4,5-BISPHOSPHATE PHOSPHODIESTERASE GAMMA 1 (EC ) (PLC-GAMMA Check to include this hit in archive report Query Observed Mr(expt) Mr(calc) Delta Miss Score Rank Peptide EDELTFTK SAIIQNVEK AQAEHMLMR (40) 1 EWYHASLTR YPINEEALEK EWYHASLTR + 1 Phospho (STY) NEPNSYAISFR AQREDELTFTK RNEPNSYAISFR LRYPINEEALEK QDGGWWRGDYGGK LSEPVPQTNAHESK IGTAEPDYGALYEGR (75) 1 IGTAEPDYGALYEGR + 1 Phospho (STY) (22) 1 IGTAEPDYGALYEGR + 1 Phospho (STY) NPGFYVEANPMPTFK (56) 1 IGTAEPDYGALYEGR + 2 Phospho (STY) (76) 1 NPGFYVEANPMPTFK + 1 Phospho (STY) ESETFVGDYTLSFWR SAIIQNVEKQDGGWWR LLTEYCIETGAPDGSFLVR LSEPVPQTNAHESKEWYHASLTR 11

12 Phosphorylation Site Mapping by DDA/ Database Searching-PLCγ IGTAEPDYGALYEGR y10 y4 Nonphos IGTAEPDpYGALpYEGR Y10* Y4* Diphos (Y448 +Y552) y4 IGTAEPDpYGALYEGR Y10* y4 y4 Monophos IGTAEPDY(448)GALY(452)EGR Two sites ID d within this peptide p All phos in monophos peptide is on Y448 Less phosphopeptide coverage as sample complexity increases 12

13 Issues with DDA Assignments Phosphorylation of Bri-1 Treated Untreated T1169 T

14 Elution profile for EIQAGSGIDSQSTIR from LC/MS E (Data Independent d Acquisition, iti DIA) Nonphos Monophos DDA 14

15 Mascot assigns phosphorylation at T1169, although the spectrum captured product ions from two species differing by phosphorylation site Treated Score 77 EIQAGSGIDSQSTIR Q 15

16 Elution profile for phospho- EIQAGSGIDSQSTIR LC/MS E independently captures both forms XFW_1_flagBRI1_CD_bak1-CD_MSE_25Mar08 XFW_1_flagBRI1_CD_bak1-CD_MSE_25Mar08 1: TOF MS ES Da 4.67e % Time 16

17 Product ions unique to each phosphorylation site S1166 S1168 T1169 T1169 S1168 S1166 b-98 b b-98 b b-98 b y y-98 y y-98 y y Arg Ile Thr Ser Gln Ser Asp Ile Gly Ser Gly Ala Gln Ile Glu Ret. Time Site S Y Y S Y Y Y Y Y T Y - S1166: 476.3, S1168: 389.3, 556.3, 458.3, , T1169: S1168/T S

18 S1166 (23.9 ) Ret. Time Site S Y Y S Y Y Y Y Y T Y - 18

19 DDA vs DIA Interpret DDA derived product ion spectra for phosphorylation site assignment cautiously Realize spectra may actually be a composite of multiple species with different sites DIA methods like MS E provide an alternative for qualitative assignment which doesn t suffer from duty cycle limitations it ti of DDA 19

20 Diagnostic Ions for Phosphorylation in Negative Ion CID Spectra pser or pthr containing peptides 79 (PO 3- ), 63 (PO 2- ), and 97 (H 2 PO 4- ) [M-H-H 3 PO 4 ] - (Loss of 98) ptyr 79 (PO ), 63 (PO 2- ) [M-H-HPO 3 ] - (Loss of 80) 20

21 Orifice Potential Stepping LC/MS -250V 79 (PO 3- ) Re elative Intensity 97 (H 2 PO 4- ) [M-2H] V [M-H] V Ding et al., 1994; Huddleston et al.,

22 Diagnostic 79 ions Yields a Chromatographic Marker for Phosphopeptides h TIC TIC SIC m/z 79 SIC m/z 79 Ding et al., Rapid Commun. Mass Spectrom.,

23 Low Mass Product Ions in Negative Ion CID 23

24 LC/MS with Stepped Orifice Potential Scanning for Phosphopeptides Perform one stepped orifice LC/MS experiment in negative ion mode. Tentative identification of phosphopeptides based on coelution with phosphopeptide marker ion and MW Perform one LC/MS experiment in positive ion mode. Confirm MW of putative phosphopeptide Perform one LC/MS/MS experiment in positive ion mode. Target putative phosphopeptides Split flow LC/MS with fraction collection Use m/z 79 ion to mark fractions containing phosphopeptides Further interrogation of phosphopeptide fractions to sequence phosphopeptides and identify site(s) of phosphorylation 24

25 Precursor Ion Scanning for m/z 79 Precursor ion scanning for m/z 79 may be used in a similar manner to stepped orifice potential Kassel et al., In Mass Spectrometry of Biological Materials, 2nd Ed. New York: Marcel Dekker, 1998, p

26 Neutral Losses, Blessing or Curse? Marker ions can be useful as a signature to flag putative phospho-peptides for further analysis However, for ps and pt containing i peptides, the primary fragmentation pathway often involves loss of the phosphate from the precursor. Resulting product ion spectra are often devoid of peptide backbone-sequence specific product ions 26

27 Neutral Losses, Blessing or Curse? 27

28 Neutral loss of phosphate can be used to trigger an additional stage of fragmentation on an ion trap 28

29 MS->MS 2 (831.6)->MS 3 (782.1) Additional Sequence Information by Fragmenting Peptide from Neutral Loss Courtesy of David Kusel, Ken Miller, ThermoFisher 29

30 ETD Provides an Alternative to CID for Improving Phosphopeptide Product Ion Spectra 11_08_2005_AKT4_ETD_load_elute01 #11690 RT: AV: 1 NL: 8.02E3 T: ITMS + c NSI d Full ms @35.00 [ ] Tubulin peptide: VRTGtYRQLFHPE MH MH _08_2005_AKT4_ETD_load_elute01 #11367 RT: AV: 1 NL: 4.31E2 T: ITMS + c NSI d Full ms @35.00 [ ] MH PLCG1 peptide: RGSDASGQLFHGRAREGsFE Relative Abundance z 3 z 4 c 5 c c 3 2 c c 8 c z c z c z 9 10 z c z m/z z10 10 z 11 z 12 Relative Abundance c c c 10 c 3 z 10 z 17 z c 7 c 8 c z c 4 c c z 14 z7 9 z 8 z 11 6 c z c c14 z 13 z c 17 c m/z T: ITMS + c NSI d Full m s @ [ ] 11_08_2005_akt4_etd_load_elute02 #13021 RT: AV: MH +2 1 NL: 7.26E4 Relative Abundance e S6 peptide: QIAKRRRLSsLRASTSKSE z 3 z 35 c 4 c c c z z 6 z 7 c z z 9 c c c z 4 c 3 z c c 10 c 11 z z c c 14 z z m/z Courtesy of David Kusel, Ken Miller, ThermoFisher Relative Abundance FLNA peptide: NGATGPVKRAREEtDKEEPASKQQKTE c 4 z 25 3 z c z z 7 z z 13 c z 12 c c c c z c c c c z z z c c m/z z 16

31 ETD Provides Complementary Phosphopeptide Coverage Compared to CID MS 2 /MS peptides MSA ETD 531 peptides MS2 / MS3 ETD Number of phosphopeptides average peptide length average number of basic residues R s (%) R t (%) Overlap MS 2 /MS 3 : ETD 0.9% MS 2 /MS 3 : MSA ~60-70% K s (%) K t (%) RR..s/t (%) R.R..s/t (%) Courtesy of David Kusel, Ken Miller, ThermoFisher 31

32 Affinity Isolation of Phoshopeptides p Anion Exchange Phospho-antibody h affinity it IMAC (Immobilized Metal-ion Affinity Chromatography) TiO 2 affinity Other Common Challenges Specificity Efficiency of capture Peptide/protein/sample dependent!!! Common requirement: SAMPLE MUST BE IN AN APPROPRIATE STATE FOR CAPTURE (ph, buffer, salt concentration) 32

33 Affinity Isolation of Phoshopeptides IMAC: Immobilized Metal ion Affinity Chromatography Fe 3+ -IDA (iminodiacetic acid) Fe 3+ 33

34 Affinity Isolation of Phoshopeptides p Metal Ions Fe 3+ Ga 3+ Loading Buffers 01%A 0.1% Acetic Acid 50mM 2-(N-morpholino)-ethanesulfonic acid (MES), ph 5.5, 1M NaCl Elution Buffers AmBic, ph 8.0 Dilute ammonium hydroxide Phosphate buffer, ph 8 Many kits are commercially available with their own recipes and cocktails No magic bullets 34

35 IMAC Pipet Tip Microcolumns 1. Pack MC resin into tip (after inserting frit) using water. Do not let liquid level fall below surface of MC resin. 2. If resin is precharged with another metal, strip metal with 250 ul 100 mm EDTA, ph After washing tip with 250 ul water, charge column with 250 ul of either 20 mm GaCl 3 or 20 mm FeCl 3. Caution: GaCl 3 (s) must be dissolved slowly with stirring under a hood. HCl(g) is liberated quite violently during dissolution. 4. After washing away excess metal ion with water, equilibrate tip column with loading buffer (0.1% HOAc or 50 mm MES, ph 5.5, 1 M NaCl). 5. Dissolve sample into 200 ul of loading buffer. Sample volumes > 50 ul might require a larger volume of loading buffer. 6. Load sample onto column adjusting flow rate to ul/min. 7. Wash column with another ul of loading buffer. 8. Elute phosphopeptides with 500 mm NH 4 HCO 3, ph 8. 35

36 Ga-Sepharose-NTA Isolation of a CD19 Phosphopeptide from an Abl/CD19 Reaction Asp-N Digest not phos IMAC Eluant Esterification of carboxylate groups with MeOH can improve selectivity Ficarro et al, JBC, 2003, p

37 Chemical Modification of Phosphates for Affinity Isolation/Relative Quantitation A number of approaches have been reported where ps or pt are chemically modified with a tag to facilitate phosphopeptide isolation and/or quantitation These methods tend to be plagued with issues around recovery and side reactions Work with standards, difficulty with real samples Not broadly used For relative quantitation between treatment states, label-free methods are preferable Zhou et al. 2001, Knight et al. 2003, Adamczyk et al. 2001, Oda et al. 2001, Weckworth et al. 2000, Goshe et al. 2001, Zhang et al

38 PhIAT (Phosphoprotein Isotope-coded Affinity Tag) Goshe et al. 2001, Anal. Chem. vol. 73, no. 11, pp

39 Phosphorylation Site Specific Quantitation Determining Phosphorylation Stoichiometry MS response of phosphorylated and nonphosphorylated forms of a given peptide are roughly equivalent and can be used to semiquantitate site specific phosphorylation stoichiometry (+/- 30%, Carr et al, Anal. Biochem., 1996, 68, p.1). Calculate % phosphorylated based on total peak area (phosphorylated + nonphosphorylated intensities) within a sample Calculate difference between treatments 39

40 Combining LC/MS/MS and LC/MS for Site Specific Quantitation 1. P10686 Mass: Total score: 873 Peptides matched: 52 PIP4_RAT 1-PHOSPHATIDYLINOSITOL-4,5-BISPHOSPHATE PHOSPHODIESTERASE GAMMA 1 (EC ) (PLC-GAMMA Check to include this hit in archive report Query Observed Mr(expt) Mr(calc) Delta Miss Score Rank Peptide EDELTFTK SAIIQNVEK AQAEHMLMR (40) 1 EWYHASLTR YPINEEALEK EWYHASLTR + 1 Phospho (STY) NEPNSYAISFR AQREDELTFTK RNEPNSYAISFR LRYPINEEALEK QDGGWWRGDYGGK LSEPVPQTNAHESK IGTAEPDYGALYEGR (75) 1 IGTAEPDYGALYEGR + 1 Phospho (STY) (22) 1 IGTAEPDYGALYEGR + 1 Phospho (STY) NPGFYVEANPMPTFK (56) 1 IGTAEPDYGALYEGR + 2 Phospho (STY) (76) 1 NPGFYVEANPMPTFK + 1 Phospho (STY) ESETFVGDYTLSFWR SAIIQNVEKQDGGWWR LLTEYCIETGAPDGSFLVR LSEPVPQTNAHESKEWYHASLTR Mascot Matches for PLCγ Peptide EWpYHASLTR EWYHASLTR y7 EWpYHASLTR y7 Nonphos Monophos 40

41 Phosphorylation Stoichiometry for PLCγ Peptide EWpYHASLTR Phos % Phos = 5.5/( )*100 = 0.6% Phos NonPhos 41

42 Phosphorylation Site Stoichiometry by LC/MS E Bri1 Treated vs Untreated 42

43 Bri1 treated (S1166) Nonphos: Phos: Calculation %Phos = (Intensity Phos/Total Intensity) * 100% %Phos = [132628/( )] * 100% % Phos = 30% 43

44 Phosphorylation Site Quantitation Summary EIQAGSGIDSQSTIR Treated + Bak1 Untreated - Bak1 S % 18% S1168/T % 22% Conclusion: S1166, but not S1168/T1169, is responsive to treatment. 44

45 PhosphoProteomics Isolation and characterization of phosphorylation at proteome level Apply methods discussed earlier on a larger scale of complexity 2,002 phos sites in 967 proteins 45