Protein Phosphorylation

Phosphorylation Site Mapping Phosphorylation Site Mapping

1

Protein Phosphorylation

• Reversible protein phosphorylation is the most common signal transduction event.

• Kinases catalyze the transfer of a Kinases catalyze the transfer of a γ phosphate group from ATP 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.

A 1/3 f t i th ht t b h h l t d

• As many as 1/3 of proteins thought to be phosphorylated.

• An estimated 2-5% of all proteins thought to be kinases.

• Relative abundance of phosphoresidues:

pSer: 90% pThr: 10% pTyr: 0 05%

– pSer: 90% pThr: 10% pTyr: 0.05%

2

Phosphorylation Analysis: The Relevant Phosphorylation Analysis: The Relevant

Questions

• How heavily phosphorylated is the protein? How heavily phosphorylated is the protein?

(stoichiometry)

• On which residues is the protein phosphorylated?

(site identification) (site identification)

• How heavily is each site phosphorylated? (site- specific stoichiometry)

• Phosphoproteomics: How does the

• Phosphoproteomics: How does the

phosphoproteome change in response to a stimulus?

• Challenges

low levels of phosphorylated proteins – low levels of phosphorylated proteins – low phosphorylation stoichiometry – transient modification

3

Phosphorylation Analysis:

The Toolbox

• Affinity Purification Methods

• Affinity Purification Methods

– Phosphoantibody, IMAC, TiO ₂ , SAX

ESI/MALDI MS

• ESI/MALDI-MS

• Tandem MS

CID ETD k i d /

– CID, ETD, marker ions product/precursor ion scanning

• Chemical Labeling Methods

• Chemical Labeling Methods

– Isolation, relative quantitation

4

Phosphorylation Stoichiometry from an Intact Protein Molecular Weight (ESI) Intact Protein Molecular Weight (ESI)

CFM-S T90 Hassell, A 100

q3t00134 443 (8.482) Sm (Mn, 3x5.00); Cm (437:451) TOF MS ES+

1048.64 100

917.65

CFM-S T90 Hassell, A 100

q3t00134 443 (8.482) M1 [Ev-65849,It9] (Gs,0.750,1378:1898,2.00,L40,R20); Sm (Mn, 3x5.00);

37606.00 95.2 37526.00

37368.00

37688.00

Recombinant Kinase

1313.98

37288.00

37448.00

37768.00

Recombinant Kinase Autophosphorylation

%

893.53

873.51

% 37208.00

584.31

580.36

780.44

752.45

1332.81 1390.90

1443.40 1447.51

37166.00

Moles Phosphate/Mole Protein

0 1 2 3 4 5 6

500 750 1000 1250 1500 1750 2000 2250

m/z 0

553.39 1505.32

37000 37200 37400 37600 37800

mass 0

p

80 Da Spacing/Phos

5

Need purified protein; more of a challenge by MALDI-Tof to

resolve the 80Da difference

Mapping of Phosphorylation Sites

Sample protein or proteome

Digest

M k I

Isolate pPeptides

MS Analysis

IMAC, TiO ₂ SAX, etc.

MALDI,

Marker Ions, Neutral Losses, Peptide Sequence

Identify pPeptides/

MS Analysis

LC/MS, LC/MS/MS Marker Ions,

Neutral Losses, Peptide Sequence

Assign Site y

Id tif P tid /

Workflow taken depends

Peptide Sequence

6

Identify pPeptides/

Assign Site

upon tools, sample,

coverage requirements

Mapping of Phosphorylation Sites

• Phosphoprotein is cleaved (digested) with a protease.

p

• Use of multiple proteases of differing specificity can increase coverage of the protein sequence.

K P P COOH

Cleavage with Trypsin

H ₂ N

R COOH

R K

K Y Y K K Y K

Y

Cleavage with Asp-N

H ₂ N

COOH Y

P

Y P

Y D Y

D

D

D D

+80 Da mass shift per phosphate group added 7

Metastable Decomposition

f S S

of pS and pT in MALDI-MS

Abundant Ions Observed Include:

[MH-H ₃ PO ₄ ] ⁺ (or MH-98) and [MH HPO ] ⁺ (or MH 80)

[MH-HPO ₃ ] (or MH-80)

Matrix effects: αHCA vs DHB

8

Annan et al., Anal. Chem., 1996, 68, 3413.

Metastable Decomposition of pY in MALDI-MS p p

For pTyr containing peptides:

[MH-H ₃ PO ₄ ] ⁺ <<< than for pSer or pThr (MH 98)

pSer or pThr (MH-98)

[MH-HPO ₃ ] ⁺ < than for pSer or pThr (MH-80)

or pThr (MH 80)

Can also analyze digests/

fractions before and after treatment with Calf Intestinal

Annan et al., Anal. Chem., 1996, 68, 3413. 9

Phosphatase (-80Da shift

with removal of phosphate

group)

Phosphorylation Site Mapping by Data Di t d A i iti (DDA)

Directed Acquisition (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

10

– Limitations of DDA

– Poor quality CID spectra for some pS/pT peptides

Phosphorylation Site Mapping by DDA/Database Searching-

Abl Phosphorylation of PLCγ

• Sample: GST-PLCγ/Abl kinase reaction mix

1. P10686 Mass: 148454 Total score: 873 Peptides matched: 52

PIP4_RAT 1-PHOSPHATIDYLINOSITOL-4,5-BISPHOSPHATE PHOSPHODIESTERASE GAMMA 1 (EC 3.1.4.11) (PLC-GAMMA

Check to include this hit in archive report

Query Observed Mr(expt) Mr(calc) Delta Miss Score Rank Peptide

• Analysis by data directed LC/MS/MS

P d t i t

43 491.67 981.32 981.47 -0.15 0 42 1 EDELTFTK 48 501.18 1000.34 1000.56 -0.21 0 50 1 SAIIQNVEK 75 543.66 1085.31 1085.51 -0.20 0 52 1 AQAEHMLMR 90 581.68 1161.35 1161.56 -0.21 0 (40) 1 EWYHASLTR 102 603.21 1204.41 1204.60 -0.19 0 63 1 YPINEEALEK

109 621.65 1241.29 1241.52 -0.23 0 47 1 EWYHASLTR + 1 Phospho (STY) 116 649.20 1296.39 1296.61 -0.22 0 48 1 NEPNSYAISFR

127 446 50 1336 47 1336 66 0 19 1 52 1 AQREDELTFTK

• Product ion spectra searched with variable modification for

127 446.50 1336.47 1336.66 -0.19 1 52 1 AQREDELTFTK 141 727.22 1452.43 1452.71 -0.28 1 13 3 RNEPNSYAISFR 143 737.76 1473.51 1473.78 -0.28 1 57 1 LRYPINEEALEK 156 494.48 1480.41 1480.65 -0.24 1 12 2 QDGGWWRGDYGGK 165 768.74 1535.45 1535.76 -0.30 0 61 1 LSEPVPQTNAHESK 186 805.68 1609.35 1610.76 -1.41 0 101 1 IGTAEPDYGALYEGR

206 846.20 1690.38 1690.72 -0.34 0 (75) 1 IGTAEPDYGALYEGR + 1 Phospho (STY) 208 846.24 1690.47 1690.72 -0.26 0 (22) 1 IGTAEPDYGALYEGR + 1 Phospho (STY)

phosphorylation

• 4 Phosphorylation sites tentatively ID’d

( ) p ( )

209 856.25 1710.48 1710.81 -0.33 0 102 1 NPGFYVEANPMPTFK

216 886.20 1770.38 1770.69 -0.31 0 (56) 1 IGTAEPDYGALYEGR + 2 Phospho (STY) 217 896.23 1790.44 1790.77 -0.34 0 (76) 1 NPGFYVEANPMPTFK + 1 Phospho (STY) 230 918.78 1835.54 1835.84 -0.29 0 36 1 ESETFVGDYTLSFWR

234 629.54 1885.59 1885.94 -0.36 1 58 1 SAIIQNVEKQDGGWWR 239 695.22 2082.65 2083.03 -0.38 0 61 1 LLTEYCIETGAPDGSFLVR 253 670.70 2678.77 2679.30 -0.53 1 31 1 LSEPVPQTNAHESKEWYHASLTR

11

tentatively ID d

Phosphorylation Site Mapping by DDA/

Database Searching PLCγ Database Searching-PLCγ

IGTAEPDYGALYEGR Nonphos

y4 y10

Diphos (Y448 +Y552) IGTAEPDpYGALpYEGR

Y4*

Y10*

y4

y y

IGTAEPDpYGALYEGR

y4 Y10*

Monophos

• Two sites ID’d within this peptide

IGTAEPDY(448)GAL Y(452)EGR

y4

p p

• All phos in monophos peptide is on Y448

12

• Less phosphopeptide coverage as sample complexity increases

Issues with DDA Assignments Phosphorylation of Bri 1

Phosphorylation of Bri-1

Treated Untreated

T1169 T1169 13

Elution profile for EIQAGSGIDSQSTIR from LC/MS ^E (D t I d d t A i iti DIA) LC/MS ^E (Data Independent Acquisition, DIA)

Nonphos Nonphos

Monophos

DDA DDA

14

Mascot assigns phosphorylation at T1169,

although the spectrum captured product ions from g p p p two species differing by phosphorylation site

EIQAGSGIDSQSTIR

Treated Score 77

Q Q

15

Elution profile for phospho- EIQAGSGIDSQSTIR

EIQAGSGIDSQSTIR

LC/MS ^E independently captures both forms

XFW_1_flagBRI1_CD_bak1-CD_MSE_25Mar08

XFW 1 fl BRI1 CD b k1 CD MSE 25M 08 1 TOF MS ES+

100

XFW_1_flagBRI1_CD_bak1-CD_MSE_25Mar08 1: TOF MS ES+

821.37 0.10Da 4.67e3

23.96

23.42

%

Time

20.50 21.00 21.50 22.00 22.50 23.00 23.50 24.00 24.50 25.00 25.50 26.00 26.50 27.00 27.50

0

16

Product ions unique to each phosphorylation site

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-98

15 - - - - - - Arg 175.1 175.1 175.1 1

14 1369.6 1467.6 1369.6 1467.6 1369.6 1467.6 Ile 288.2 288.2 288.2 2

13 1256 6 1354 6 1256 6 1354 6 1256 6 1354 6 Th 469 2 371 2 389 3 389 3 3

13 1256.6 1354.6 1256.6 1354.6 1256.6 1354.6 Thr 469.2 371.2 389.3 389.3 3

12 1155.5 1253.5 1155.5 1253.5 1173.5 Ser 556.3 458.3 556.3 458.3 476.3 4

11 1068.5 1166.5 1086.5 1086.5 Gln 684.3 586.3 684.3 586.3 604.3 5

10 940.4 1038.4 958.5 958.5 Ser 771.3 673.3 771.3 673.3 771.3 673.3 6

9 871.4 871.4 871.4 Asp 886.7 788.7 886.7 788.7 886.7 788.7 7

8 756.4 756.4 756.4 Ile 999.5 901.5 999.5 901.5 999.5 901.5 8

7 643.3 643.3 643.3 Gly y 1056.5 958.5 1056.5 958.5 1056.5 958.5 9

6 586.3 586.3 586.3 Ser 1143.5 1045.5 1143.5 1045.5 1143.5 1045.5 10

5 499.3 499.3 499.3 Gly 1200.5 1102.5 1200.5 1102.5 1200.5 1102.5 11

4 442.2 442.2 442.2 Ala 1271.6 1173.6 1271.6 1173.6 1271.6 1173.6 12

3 371.2 371.2 371.2 Gln 1399.6 1301.6 1399.6 1301.6 1399.6 1301.6 13

2 243.1 243.1 243.1 Ile 1512.7 1414.7 1512.7 1414.7 1512.7 1414.7 14

1 130.1 130.1 130.1 Glu - - - - - - 15

S1166: 476.3, 604.3

S1168: 389.3, 556.3, 458.3, 1166.5, 1068.5 T1169: 469.2

Ret. Time

Site 23.4 23.9

S1166

476.3 - Y

S1166 23.9’

23 9’

604.3 - Y

S1168

389.3 - Y

556.3 Y Y

458.3 Y -

1166 5

S1168/T1169 23.9’

17

1166.5 - -

1068.5 - Y

T1169

469.2 Y -

S1166 (23.9’)

Ret. Time

Site 23.4 23.9

S1166

476.3 - Y

604 3 - Y

604.3 Y

S1168

389.3 - Y

556.3 Y Y

458.3 Y -

1166.5 - -

1068.5 - Y

T1169

469.2 Y -

18

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

ff f d t l li it ti f DDA suffer from duty cycle limitations of DDA

19

Diagnostic Ions for Phosphorylation Diagnostic Ions for Phosphorylation

in Negative Ion CID Spectra

• pSer or pThr containing peptides

– 79 (PO 79 (PO _{3 3} ^- ), 63 (PO ), 63 (PO _{2 2} ^- ), and 97 (H ), and 97 (H _{2 2} PO PO _{4 4} ^- ) ) – [M-H-H ₃ PO ₄ ] ^- (Loss of 98)

• pTyr

79 (PO ^- ) 63 (PO ^- ) – 79 (PO ₃ ^- ), 63 (PO ₂ ^- )

– [M-H-HPO ₃ ] ^- (Loss of 80)

20

Orifice Potential Stepping LC/MS pp g

y

79 (PO ₃ ^- )

[M 2H] ^2- -250V

140V

e lative Intensit y

97 (H ₂ PO ₄ ^- )

[M-2H] ²

[M H]

80V

-140V

200 400 600 800 1000 1200

R e

[M-H] ^- -80V

200 400 600 800 1000 1200

Ding et al., 1994; Huddleston et al., 1994 21

Diagnostic 79 ions Yields a Chromatographic

M k f Ph h tid

Marker for Phosphopeptides

TIC TIC

SIC m/z 79

SIC m/z 79 SIC m/z 79

Ding et al., Rapid Commun. Mass Spectrom., 1994 22

Low Mass Product Ions in Negative Ion CID Low Mass Product Ions in Negative Ion CID

23

LC/MS with Stepped Orifice Potential 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

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

• 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

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

25 Kassel et al., In Mass Spectrometry of Biological Materials, 2nd Ed.

New York: Marcel Dekker, 1998, p. 137-158.

Neutral Losses Blessing or Curse?

Neutral Losses, Blessing or Curse?

• Marker ions can be useful as a signature to g flag putative phospho-peptides for further analysis

H f S d T i i id

• However, for pS and pT containing peptides, the primary fragmentation pathway often

involves loss of the phosphate from the involves loss of the phosphate from the precursor.

• Resulting product ion spectra are often devoid of peptide backbone-sequence specific product ions

26

Neutral Losses Blessing or Curse?

Neutral Losses, Blessing or Curse?

27

Neutral loss of phosphate can be used to trigger an additional stage of used to trigger an additional stage of

fragmentation on an ion trap

28

MS->MS ² (831.6)->MS ³ (782.1) Additional Sequence Information by Additional Sequence Information by Fragmenting Peptide from Neutral Loss

29

Courtesy of David Kusel, Ken Miller, ThermoFisher

ETD Provides an Alternative to CID for Improving Phosphopeptide Product Ion Spectra

Phosphopeptide Product Ion Spectra

1 1 _ 0 8 _ 2 0 0 5 _ A K T4 _ E TD _ lo a d _ e lute 0 1 #1 1 3 6 7 R T:8 2 .1 2 A V :1 N L :4 .3 1 E 2 T:ITM S + c N S I d F ull m s 2 5 6 2 .4 7 @ 3 5 .0 0 [ 5 0 .0 0 -2 0 0 0 .0 0 ]

7 0 7 5 8 0 8 5 9 0 9 5

1 0 0

MH

1 1 2 3 .2 8₂⁺²

1 1 _ 0 8 _ 2 0 0 5 _ A K T4 _ E TD _ lo a d _ e lute 0 1 #1 1 3 6 7 R T:8 2 .1 2 A V :1 N L :4 .3 1 E 2 T:ITM S + c N S I d F ull m s 2 5 6 2 .4 7 @ 3 5 .0 0 [ 5 0 .0 0 -2 0 0 0 .0 0 ]

7 0 7 5 8 0 8 5 9 0 9 5

1 0 0

MH

1 1 2 3 .2 8₂⁺²

1 1 _ 0 8 _ 2 0 0 5 _ A K T4 _ E TD _ lo a d _ e lute 0 1 #1 1 3 6 7 R T:8 2 .1 2 A V :1 N L :4 .3 1 E 2 T:ITM S + c N S I d F ull m s 2 5 6 2 .4 7 @ 3 5 .0 0 [ 5 0 .0 0 -2 0 0 0 .0 0 ]

7 0 7 5 8 0 8 5 9 0 9 5

1 0 0

MH

1 1 2 3 .2 8₂⁺²

PLCG1 peptide: RGSDASGQLFHGRAREGsFE

1 1 _ 0 8 _ 2 0 0 5 _ A K T4 _ E TD _ lo a d _ e lute 0 1 #1 1 6 9 0R T:8 4 .4 4 A V :1 N L :8 .0 2 E 3 T:ITM S + c N S I d F ull m s 2 5 6 2 .1 7 @ 3 5 .0 0 [ 5 0 .0 0 -2 0 0 0 .0 0 ]

7 0 7 5 8 0 8 5 9 0 9 5

1 0 0 1 6 8 4 .6 8

8 4 2 .6 7

MH

₂⁺²

MH

⁺¹

1 1 _ 0 8 _ 2 0 0 5 _ A K T4 _ E TD _ lo a d _ e lute 0 1 #1 1 6 9 0R T:8 4 .4 4 A V :1 N L :8 .0 2 E 3 T:ITM S + c N S I d F ull m s 2 5 6 2 .1 7 @ 3 5 .0 0 [ 5 0 .0 0 -2 0 0 0 .0 0 ]

7 0 7 5 8 0 8 5 9 0 9 5

1 0 0 1 6 8 4 .6 8

8 4 2 .6 7

MH

₂⁺²

MH

⁺¹

1 1 _ 0 8 _ 2 0 0 5 _ A K T4 _ E TD _ lo a d _ e lute 0 1 #1 1 6 9 0R T:8 4 .4 4 A V :1 N L :8 .0 2 E 3 T:ITM S + c N S I d F ull m s 2 5 6 2 .1 7 @ 3 5 .0 0 [ 5 0 .0 0 -2 0 0 0 .0 0 ]

7 0 7 5 8 0 8 5 9 0 9 5

1 0 0 1 6 8 4 .6 8

8 4 2 .6 7

MH

₂⁺²

MH

⁺¹

Tubulin peptide: VRTGtYRQLFHPE

2 5 3 0 3 5 4 0 4 5 5 0 5 5 6 0 6 5

Relative Abundance

5 6 1 .2 0

c

₃

c

₁₀

z

₁₇

z

₁₀

2 5 3 0 3 5 4 0 4 5 5 0 5 5 6 0 6 5

Relative Abundance

5 6 1 .2 0

c

₃

c

₁₀

z

₁₇

z

₁₀

2 5 3 0 3 5 4 0 4 5 5 0 5 5 6 0 6 5

Relative Abundance

5 6 1 .2 0

c

₃

c

₁₀

z

₁₇

z

₁₀

2 5 3 0 3 5 4 0 4 5 5 0 5 5 6 0 6 5

Relative Abundance

6 1 2 .2 4

c c

₃

c

₅

z

₄

z

₃

z

₁₀

2 5 3 0 3 5 4 0 4 5 5 0 5 5 6 0 6 5

Relative Abundance

6 1 2 .2 4

c c

₃

c

₅

z

₄

z

₃

z

₁₀

2 5 3 0 3 5 4 0 4 5 5 0 5 5 6 0 6 5

Relative Abundance

6 1 2 .2 4

c c

₃

c

₅

z

₄

z

₃

z

₁₀

1 1 _ 0 8 _ 2 0 0 5 _ A K T 4 _ E T D _ lo a d _ e lu te 0 1 #9 5 7 0 R T :6 9 .0 3 A V :1 N L :4 .9 8 E 2

T :IT M S + c N S I d F u ll m s 2 6 1 4 2 1 @ 3 5 0 0 [ 5 0 0 0 2 0 0 0 0 0 ]

MH

2+2

1 1 _ 0 8 _ 2 0 0 5 _ A K T 4 _ E T D _ lo a d _ e lu te 0 1 #9 5 7 0 R T :6 9 .0 3 A V :1 N L :4 .9 8 E 2

T :IT M S + c N S I d F u ll m s 2 6 1 4 2 1 @ 3 5 0 0 [ 5 0 0 0 2 0 0 0 0 0 ]

MH

2+2

1 1 _ 0 8 _ 2 0 0 5 _ a k t4 _ e td _ lo a d _ e lu te 0 2 #1 3 0 2 1 R T :6 9 .6 0 A V :1 N L :7 .2 6 E 4

T :IT M S + c N S I d F u ll m s 2 4 5 2 .0 4 @ 3 5 .0 0 [ 5 0 .0 0 - 2 0 0 0 .0 0 ]

MH

⁺²

1 1 _ 0 8 _ 2 0 0 5 _ a k t4 _ e td _ lo a d _ e lu te 0 2 #1 3 0 2 1 R T :6 9 .6 0 A V :1 N L :7 .2 6 E 4

T :IT M S + c N S I d F u ll m s 2 4 5 2 .0 4 @ 3 5 .0 0 [ 5 0 .0 0 - 2 0 0 0 .0 0 ]

MH

⁺²

2 0 0 4 0 0 6 0 0 8 0 0 1 0 0 0 1 2 0 0 1 4 0 0 1 6 0 0 1 8 0 0 2 0 0 0

m /z 0

5 1 0 1 5

2 0 3 1 8 .1 3

1 0 5 8 .8 4

1 9 6 8 .0 9 1 6 8 1 .6 0

7 4 9 .5 4 8 5 9 .2 6 1 3 8 7 .6 1

4 3 3 .1 9 6 4 8 .2 6 9 8 4 .3 7 1 2 1 0 .5 7 1 5 9 9 .6 9 1 8 1 3 .7 9

1 7 4 .1 3

5 0 5 .0 2

c

₁

c

₂

c

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c

₄

c

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c

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c

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₁₁

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₁₀

z

₈

z

₇

z

₆

2 0 0 4 0 0 6 0 0 8 0 0 1 0 0 0 1 2 0 0 1 4 0 0 1 6 0 0 1 8 0 0 2 0 0 0

m /z 0

5 1 0 1 5

2 0 3 1 8 .1 3

1 0 5 8 .8 4

1 9 6 8 .0 9 1 6 8 1 .6 0

7 4 9 .5 4 8 5 9 .2 6 1 3 8 7 .6 1

4 3 3 .1 9 6 4 8 .2 6 9 8 4 .3 7 1 2 1 0 .5 7 1 5 9 9 .6 9 1 8 1 3 .7 9

1 7 4 .1 3

5 0 5 .0 2

c

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2 0 0 4 0 0 6 0 0 8 0 0 1 0 0 0 1 2 0 0 1 4 0 0 1 6 0 0 1 8 0 0 2 0 0 0

m /z 0

5 1 0 1 5

2 0 3 1 8 .1 3

1 0 5 8 .8 4

1 9 6 8 .0 9 1 6 8 1 .6 0

7 4 9 .5 4 8 5 9 .2 6 1 3 8 7 .6 1

4 3 3 .1 9 6 4 8 .2 6 9 8 4 .3 7 1 2 1 0 .5 7 1 5 9 9 .6 9 1 8 1 3 .7 9

1 7 4 .1 3

5 0 5 .0 2

c

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2 0 0 4 0 0 6 0 0 8 0 0 1 0 0 0 1 2 0 0 1 4 0 0 1 6 0 0 1 8 0 0 2 0 0 0

m /z 0

5 1 0 1 5

2 0 2 7 3 .2 0

1 4 1 2 .3 5 1 0 7 3 .3 6

6 2 6 .0 7

4 3 1 .3 1 9 3 1 .4 4

1 2 5 4 .2 6

1 5 6 9 .5 0

5 6 1 .1 1 1 1 7 2 .5 8

3 7 4 .2 4 1 3 1 9 .6 4

2 2 9 .2 1 6 4 2 .4 2 1 5 0 8 .7 3 1 7 4 6 .5 4 1 8 5 2 .3 2

c

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10

z

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z

₁₂

2 0 0 4 0 0 6 0 0 8 0 0 1 0 0 0 1 2 0 0 1 4 0 0 1 6 0 0 1 8 0 0 2 0 0 0

m /z 0

5 1 0 1 5

2 0 2 7 3 .2 0

1 4 1 2 .3 5 1 0 7 3 .3 6

6 2 6 .0 7

4 3 1 .3 1 9 3 1 .4 4

1 2 5 4 .2 6

1 5 6 9 .5 0

5 6 1 .1 1 1 1 7 2 .5 8

3 7 4 .2 4 1 3 1 9 .6 4

2 2 9 .2 1 6 4 2 .4 2 1 5 0 8 .7 3 1 7 4 6 .5 4 1 8 5 2 .3 2

c

₂

c

₃

c

₄

c

₆

c

₇

c

₈

c

₉

c

₁₀

z

₅

z

₆

z

₇

z

₈

z

₉

10

z

₁₁

z

₁₂

2 0 0 4 0 0 6 0 0 8 0 0 1 0 0 0 1 2 0 0 1 4 0 0 1 6 0 0 1 8 0 0 2 0 0 0

m /z 0

5 1 0 1 5

2 0 2 7 3 .2 0

1 4 1 2 .3 5 1 0 7 3 .3 6

6 2 6 .0 7

4 3 1 .3 1 9 3 1 .4 4

1 2 5 4 .2 6

1 5 6 9 .5 0

5 6 1 .1 1 1 1 7 2 .5 8

3 7 4 .2 4 1 3 1 9 .6 4

2 2 9 .2 1 6 4 2 .4 2 1 5 0 8 .7 3 1 7 4 6 .5 4 1 8 5 2 .3 2

c

₂

c

₃

c

₄

c

₆

c

₇

c

₈

c

₉

c

₁₀

z

₅

z

₆

z

₇

z

₈

z

₉

10

z

₁₁

z

₁₂

T :IT M S + c N S I d F u ll m s 2 6 1 4 .2 1 @ 3 5 .0 0 [ 5 0 .0 0 - 2 0 0 0 .0 0 ]

6 0 6 5 7 0 7 5 8 0 8 5 9 0 9 5

1 0 0 1 5 3 3 .8 3

T :IT M S + c N S I d F u ll m s 2 6 1 4 .2 1 @ 3 5 .0 0 [ 5 0 .0 0 - 2 0 0 0 .0 0 ] 2

6 0 6 5 7 0 7 5 8 0 8 5 9 0 9 5

1 0 0 1 5 3 3 .8 3

2

FLNA peptide: NGATGPVKRAREEtDKEEPASKQQKTE

6 0 6 5 7 0 7 5 8 0 8 5 9 0 9 5 1 0 0

e

1 1 2 8 .5 1

6 0 6 5 7 0 7 5 8 0 8 5 9 0 9 5 1 0 0

e

1 1 2 8 .5 1

S6 peptide: QIAKRRRLSsLRASTSKSE

2 0 2 5 3 0 3 5 4 0 4 5 5 0 5 5 6 0

Relative Abundance

1 5 1 0 .9 9 3 6 1 .2 3

6 1 3 .0 0

1 2 2 5 .2 8 1 1 3 0 .4 5 1 0 2 2 .7 3

1 5 6 4 7 1

z

₁₃

c

₃

c

4

c

7

c c c

¹³

z

₁₂

c

₁₄

z

₁₀

z

8

z

7

z

₅

z

₃

2 0 2 5 3 0 3 5 4 0 4 5 5 0 5 5 6 0

Relative Abundance

1 5 1 0 .9 9 3 6 1 .2 3

6 1 3 .0 0

1 2 2 5 .2 8 1 1 3 0 .4 5 1 0 2 2 .7 3

1 5 6 4 7 1

z

₁₃

c

₃

c

4

c

7

c c c

¹³

z

₁₂

c

₁₄

z

₁₀

z

8

z

7

z

₅

z

₃

1 5 2 0 2 5 3 0 3 5 4 0 4 5 5 0 5 5

Relative Abundance

1 0 9 8 .8 1

1 7 9 8 .8 5 6 1 4 .3 8

4 5 8 .2 4

3 4 7 .1 5

1 9 2 7 .9 0 1 6 4 2 .8 1

8 4 9 .1 9

7 7 0 .5 8 1 4 0 6 7 2 1 4 8 6 7 1

c

4

c

8

c

₉

c

₁₀

c

11

c

c

₁₆⁺²

c

₅

c

6

c c

₂

z

16

z

₁₃

z

₁₄

z

₁₅

z

9

z

₁₀

z

₂

z

₇

z

3

z

₈

z

6

1 5 2 0 2 5 3 0 3 5 4 0 4 5 5 0 5 5

Relative Abundance

1 0 9 8 .8 1

1 7 9 8 .8 5 6 1 4 .3 8

4 5 8 .2 4

3 4 7 .1 5

1 9 2 7 .9 0 1 6 4 2 .8 1

8 4 9 .1 9

7 7 0 .5 8 1 4 0 6 7 2 1 4 8 6 7 1

c

4

c

8

c

₉

c

₁₀

c

11

c

c

₁₆⁺²

c

₅

c

6

c c

₂

z

16

z

₁₃

z

₁₄

z

₁₅

z

9

z

₁₀

z

₂

z

₇

z

3

z

₈

z

6

30

2 0 0 4 0 0 6 0 0 8 0 0 1 0 0 0 1 2 0 0 1 4 0 0 1 6 0 0 1 8 0 0 2 0 0 0

m /z 0

5 1 0

1 5 1 5 6 4 .7 1

2 6 0 .0 4 7 4 2 .4 5 1 4 0 3 .9 3

1 8 1 4 .6 3 9 6 9 .6 3

1 9 3 7 .5 6 1 3 5 4 .7 8

8 9 8 .6 1 4 8 9 .0 4

1 8 8 .9 8 5 7 0 .3 9 6 9 8 .3 9 8 3 2 .5 0 1 6 7 9 .8 7

1 8 0 .1 1 3 7 7 .3 3 1 7 3 6 .6 7

z

16

z

₁₄

z

₁₅

c

2

c

₃

c

8

c

9

c

₁₀

c

11

c

¹²

c

c

₁₅

c

₁₇

z

11

z

4

2 0 0 4 0 0 6 0 0 8 0 0 1 0 0 0 1 2 0 0 1 4 0 0 1 6 0 0 1 8 0 0 2 0 0 0

m /z 0

5 1 0

1 5 1 5 6 4 .7 1

2 6 0 .0 4 7 4 2 .4 5 1 4 0 3 .9 3

1 8 1 4 .6 3 9 6 9 .6 3

1 9 3 7 .5 6 1 3 5 4 .7 8

8 9 8 .6 1 4 8 9 .0 4

1 8 8 .9 8 5 7 0 .3 9 6 9 8 .3 9 8 3 2 .5 0 1 6 7 9 .8 7

1 8 0 .1 1 3 7 7 .3 3 1 7 3 6 .6 7

z

16

z

₁₄

z

₁₅

c

2

c

₃

c

8

c

9

c

₁₀

c

11

c

¹²

c

c

₁₅

c

₁₇

z

11

z

4

2 0 0 4 0 0 6 0 0 8 0 0 1 0 0 0 1 2 0 0 1 4 0 0 1 6 0 0 1 8 0 0 2 0 0 0

m /z 0

5 1 0

1 5 9 5 5 .3 3 1 4 0 6 .7 2 1 4 8 6 .7 1

1 5 6 2 .9 6 2 5 8 .9 8

1 4 6 .1 6 4 3 4 .2 3 5 6 4 .7 0 1 2 9 5 .6 0

1 2 7 8 .8 8 1 7 2 0 .9 4

c

7

8

c

₁₂

c

14

c

1

c

₂

c

₃

z

4

z

₅

6

2 0 0 4 0 0 6 0 0 8 0 0 1 0 0 0 1 2 0 0 1 4 0 0 1 6 0 0 1 8 0 0 2 0 0 0

m /z 0

5 1 0

1 5 9 5 5 .3 3 1 4 0 6 .7 2 1 4 8 6 .7 1

1 5 6 2 .9 6 2 5 8 .9 8

1 4 6 .1 6 4 3 4 .2 3 5 6 4 .7 0 1 2 9 5 .6 0

1 2 7 8 .8 8 1 7 2 0 .9 4

c

7

8

c

₁₂

c

14

c

1

c

₂

c

₃

z

4

z

₅

6

Courtesy of David Kusel, Ken Miller, ThermoFisher

ETD Provides Complementary Phosphopeptide Coverage Compared to CID

Coverage Compared to CID

ETD ETD

MS2 / MS3

ETD ETD

531 peptides MS ² /MS ³

ETD

531 peptides MS ² /MS ³

MS3

Number of phosphopeptides 127 531 average peptide length 14.4 19.5

MSA 127 peptides

MSA 127 peptides

average number of basic residues

1.7 5.2

R…s (%) 70.9 76.3

Overlap MS ² /MS ³ : ETD 0.9%

MS ² /MS ³ MSA 60 70%

Overlap MS ² /MS ³ : ETD 0.9%

MS ² /MS ³ MSA 60 70%

R…t (%) 26.0 26.6

K…s (%) 1.6 0.8

K…t (%) 2.4 0.4

MS ² /MS ³ : MSA ~60-70%

MS ² /MS ³ : MSA ~60-70%

RR..s/t (%) 6.3 41.6

R.R..s/t (%) 7.9 34.1

31

Courtesy of David Kusel, Ken Miller, ThermoFisher

Affinity Isolation of Phoshopeptides y p p

• Anion Exchange

Ph h tib d ffi it

• Phospho-antibody affinity

• IMAC (Immobilized Metal-ion Affinity Chromatography)

• TiO ₂ affinity

• Other…

• Common Challenges – Specificity

Efficiency of capture – Efficiency of capture

– Peptide/protein/sample dependent!!!

• Common requirement: SAMPLE MUST BE IN AN APPROPRIATE STATE FOR CAPTURE (pH b ffer

32

APPROPRIATE STATE FOR CAPTURE (pH, buffer,

salt concentration)

Affinity Isolation of Phoshopeptides

IMAC: Immobilized Metal ion Affinity Chromatography

Fe 3+

-IDA (iminodiacetic acid)

Fe ³⁺

33

Affinity Isolation of Phoshopeptides y p p

• Metal Ions – Fe Fe ³⁺

– Ga ³⁺

• Loading Buffers

0 1% A ti A id – 0.1% Acetic Acid

– 50mM 2-(N-morpholino)-ethanesulfonic acid (MES), pH 5.5, 1M NaCl

El ti B ff

• Elution Buffers – AmBic, pH 8.0

– Dilute ammonium hydroxide y – Phosphate buffer, pH 8

• Many kits are commercially available with their own recipes and cocktails

34

recipes and cocktails

• No magic bullets

IMAC Pipet Tip Microcolumns p p

1. Pack MC resin into tip (after inserting frit) using water Do not let liquid level fall below 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 8.5.

3. After washing tip with 250 uL water, charge l ith 250 L f ith 20 M G Cl column with 250 uL of either 20 mM GaCl3 or 20 mM FeCl3. Caution: GaCl3(s) must be dissolved slowly with stirring under a hood.

HCl(g) is liberated quite violently during dissolution

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 Sample volumes > 50 uL might require a larger volume of loading buffer.

6. Load sample onto column adjusting flow rate to 50-100 uL/min.

7. Wash column with another 250-500 uL of loading buffer

35 buffer.

8. Elute phosphopeptides with 500 mM NH4HCO3,

pH 8.

Ga-Sepharose-NTA Isolation of a CD19 Phosphopeptide from an Abl/CD19 Reaction Phosphopeptide from an Abl/CD19 Reaction

Asp-N Digest

IMAC Eluant

not phos

Esterification of carboxylate groups with MeOH can improve selectivity 36

Ficarro et al, JBC, 2003, p11579.

Chemical Modification of Phosphates for Affinity Isolation/Relative Quantitation Affinity Isolation/Relative Quantitation

• A number of approaches have been reported where pS or pT are chemically modified with a tag to

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

around recovery and side reactions

– Work with standards, difficulty with real samples

• Not broadly used

For relative quantitation between treatment states

• 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

• 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. 2002

37

PhIAT (Phosphoprotein Isotope-coded Affinity Tag)

Affinity Tag)

Goshe et al. 2001, Anal. Chem. vol. 73, no. 11, pp. 2578-2586. 38

Phosphorylation Site Specific Quantitation 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 semi

roughly equivalent and can be used to semi-

quantitate 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 within a sample

• Calculate difference between treatments

39

Combining LC/MS/MS and LC/MS for Site Specific Quantitation

Specific Quantitation

EWYHASLTR Nonphos

1. P10686 Mass: 148454 Total score: 873 Peptides matched: 52

PIP4_RAT 1-PHOSPHATIDYLINOSITOL-4,5-BISPHOSPHATE PHOSPHODIESTERASE GAMMA 1 (EC 3.1.4.11) (PLC-GAMMA y7

Check to include this hit in archive report

Query Observed Mr(expt) Mr(calc) Delta Miss Score Rank Peptide 43 491.67 981.32 981.47 -0.15 0 42 1 EDELTFTK 48 501.18 1000.34 1000.56 -0.21 0 50 1 SAIIQNVEK 75 543.66 1085.31 1085.51 -0.20 0 52 1 AQAEHMLMR 90 581.68 1161.35 1161.56 -0.21 0 (40) 1 EWYHASLTR

102 603 21 1204 41 1204 60 0 19 0 63 1 YPINEEALEK 102 603.21 1204.41 1204.60 -0.19 0 63 1 YPINEEALEK

109 621.65 1241.29 1241.52 -0.23 0 47 1 EWYHASLTR + 1 Phospho (STY) 116 649.20 1296.39 1296.61 -0.22 0 48 1 NEPNSYAISFR

127 446.50 1336.47 1336.66 -0.19 1 52 1 AQREDELTFTK 141 727.22 1452.43 1452.71 -0.28 1 13 3 RNEPNSYAISFR 143 737.76 1473.51 1473.78 -0.28 1 57 1 LRYPINEEALEK 156 494.48 1480.41 1480.65 -0.24 1 12 2 QDGGWWRGDYGGK 165 768.74 1535.45 1535.76 -0.30 0 61 1 LSEPVPQTNAHESK 186 805.68 1609.35 1610.76 -1.41 0 101 1 IGTAEPDYGALYEGR

EWpYHASLTR

y7

Monophos

206 846.20 1690.38 1690.72 -0.34 0 (75) 1 IGTAEPDYGALYEGR + 1 Phospho (STY) 208 846.24 1690.47 1690.72 -0.26 0 (22) 1 IGTAEPDYGALYEGR + 1 Phospho (STY) 209 856.25 1710.48 1710.81 -0.33 0 102 1 NPGFYVEANPMPTFK

216 886.20 1770.38 1770.69 -0.31 0 (56) 1 IGTAEPDYGALYEGR + 2 Phospho (STY) 217 896.23 1790.44 1790.77 -0.34 0 (76) 1 NPGFYVEANPMPTFK + 1 Phospho (STY) 230 918.78 1835.54 1835.84 -0.29 0 36 1 ESETFVGDYTLSFWR

234 629.54 1885.59 1885.94 -0.36 1 58 1 SAIIQNVEKQDGGWWR 239 695.22 2082.65 2083.03 -0.38 0 61 1 LLTEYCIETGAPDGSFLVR

253 670 70 2678 77 2679 30 0 53 1 31 1 LSEPVPQTNAHESKEWYHASLTR 253 670.70 2678.77 2679.30 -0.53 1 31 1 LSEPVPQTNAHESKEWYHASLTR

Mascot Matches for PLCγ Peptide EWpYHASLTR

40

Phosphorylation Stoichiometry for PLCγ Peptide EWpYHASLTR p p

Phos % Phos = 5.5/(939.8+5.5)*100

= 0.6% Phos

NonPhos NonPhos

41

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

42

Calculation

Bri1 treated (S1166) Bri1 treated (S1166) Nonphos: 310525 p Phos: 132628

%Phos = (Intensity Phos/Total Intensity) * 100%

%Phos = [132628/(132628 + 310525)] * 100%

%Phos = [132628/(132628 + 310525)] * 100%

% Phos = 30%

43

% Phos 30%

Phosphorylation Site Quantitation Phosphorylation Site Quantitation

Summary EIQAGSGIDSQSTIR + Bak1 - Bak1

Treated Untreated

S1166 30% 18%

S1168/T1169 23% 22%

S1168/T1169 23% 22%

44

Conclusion: S1166, but not S1168/T1169, is responsive

to treatment.

PhosphoProteomics p

• Isolation and characterization of phosphorylation at proteome level phosphorylation at proteome level

• Apply methods discussed earlier on a

l l f l it

larger scale of complexity

2 002 phos sites in 2,002 phos sites in 967 proteins

45