Protein and PTM Identification

IDeA National ResourceProteomics Workshop

May 24, 2017

Samuel G. Mackintosh, Ph.D.Dept. of Biochemistry and Molecular Biology University of Arkansas for Medical Sciences

sgmackintosh@uams.edu

mailto:sgmackintosh@uams.edu

Protein identification by LC-MS/MS:

• in-gel trypsin digestion• reverse-phase HPLC separation• electrospray ionization• peptide ion detection by MS• peptide fragmentation by MS/MS• protein identification by database searching

+NH3-AAGTYCAGDVR-COO-+NH3-GTHLAAECVPK-COO-

+NH3-ILESTCVYLAGR-COO-trypsin

+NH3-TYAAGDFPILTNMCR-COO-

BSA_060908_01 #647 RT: 10.21 AV: 1 NL: 2.71E3T: ITMS + c ESI d Full ms2 722.65@cid35.00 [185.00-2000.00]

200 400 600 800 1000 1200 1400 1600 1800 2000m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Ab

unda

nce

584.57

1167.49

1007.48321.27

713.57575.60 1123.34892.47249.19 437.121210.43

1730.571367.54

BSA_060908_01 #659 RT: 10.41 AV: 1 NL: 3.02E5T: ITMS + c ESI Full ms [375.00-1500.00]

400 500 600 700 800 900 1000 1100 1200 1300 1400 1500m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Ab

unda

nce

722.53

445.11 724.02500.14767.82535.89 819.97 1167.61598.73 978.95 1051.46 1365.721236.81 1448.59

B S A

[1 -6 0 7 ] m a s s = 6 9 2 9 3 .4 1 , p I = 5 .8

Cl e a v a g e a t KR

1 M K w v t f i s l l l l f s s a y s r G V F R r D T H K s e 30 31 i a h r F K d l g e e h f k G L V L I A F S Q Y L Q Q C P F 60 61 D E H V K l v n e l t e f a k T C V A D E S H A G C E K s l 90 91 h t l f g d e l c k V A S L R e t y g d m a d c c e k Q E P 120

121 E R n e c f l s h k D D S P D L P K l k p d p n t l c d e f 150 151 k A D E K k F WG K y l y e i a r R h p y f y a p e l l y y 180 181 a n k Y N G V F Q E C C Q A E D K g a c l l p k I E T M R e 210 211 k V L A S S A R q r L R c a s i q k F G E R a l k A W S V A 240 241 R l s q k F P K a e f v e v t k L V T D L T K v h k E C C H 270 271 G D L L E C A D D R a d l a k Y I C D N Q D T I S S K l k E 300 301 C C D K P L L E K s h c i a e v e k D A I P E N L P P L T A 330 331 D F A E D K d v c k N Y Q E A K d a f l g s f l y e y s r R 360 361 h p e y a v s v l l r L A K e y e a t l e e c c a k D D P H 390 391 A C Y S T V F D K l k H L V D E P Q N L I K q n c d q f e k 420 421 L G E Y G F Q N A L I V R y t r K v p q v s t p t l v e v s 450 451 r S L G K v g t r C C T K P E S E R m p c t e d y l s l i l 480 481 n r L C V L H E K t p v s e k V T K c c t e s l v n r R P C 510 511 F S A L T P D E T Y V P K a f d e k L F T F H A D I C T L P 540 541 D T E K q i k K q t a l v e l l k H K P K a t e e q l k T V 570 571 M E N F V A F V D K c c a a d d k E A C F A V E G P K l v v 600 601 s t q t a l a 607

Tryptic digestion of BSA:• cleaves C-terminal of K or R

MW rangeof peptide products:

146.19 – 2435.82

• not all peptides will beionized and detected

• there will be gapsin sequence coverage

RT: 0.00 - 30.08

0 5 10 15 20 25 30Time (min)

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Ab

unda

nce

23.76

24.97

25.2123.41

11.07 23.1311.62 25.4022.96

10.419.64 14.736.99 27.105.442.33 16.95 28.8719.95

NL:2.89E6Base Peak F: ITMS + c ESI Full ms [375.00-1500.00] MS BSA_060908_01

LC-MS of 20 fmol of BSA

H2O/acetonitrile gradient

MS scan – determines masses of intact peptides

Ecoli_041708_01 #1310 RT: 14.94 AV: 1 NL: 8.31E4F: ITMS + c ESI Full ms [375.00-1500.00]

400 500 600 700 800 900 1000 1100 1200 1300 1400 1500m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Ab

unda

nce

494.62

515.72

881.94

577.22

462.88 605.80

728.66447.17 748.59 985.51 1039.28 1133.29 1220.99 1300.73 1437.59

Data-Dependent Acquisition (DDA)

Ecoli_041708_01 #1310 RT: 14.94 AV: 1 NL: 8.31E4F: ITMS + c ESI Full ms [375.00-1500.00]

400 500 600 700 800 900 1000 1100 1200 1300 1400 1500m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Ab

unda

nce

494.62

515.72

881.94

577.22

462.88 605.80

728.66447.17 748.59 985.51 1039.28 1133.29 1220.99 1300.73 1437.59

most intense peptide ions areselected for fragmentation

Ecoli_041708_01 #1315 RT: 15.02 AV: 1 NL: 1.44E2T: ITMS + c ESI d Full ms2 728.66@cid35.00 [190.00-2000.00]

200 400 600 800 1000 1200 1400 1600 1800 2000m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Ab

unda

nce

1209.44

614.99 718.78

984.35571.37246.12

798.33 1099.40414.301308.49854.40

247.23 1327.57

MS/MS scan – determines masses of peptide fragments

MS/MS fragmentation methods:Collision-induced dissociation (CID)is most likely to occur at peptide bonds

b ions: charge is retained on N-terminus

y ions: charge is retained on C-terminus

Electron-transfer dissociation (ETD)is more likely to occur at N-Cα bonds

c ions: charge is retained on N-terminus

z ions: charge is retained on C-terminus

High-energy collisional dissociation (HCD)produces b and y ions

a/x ions

c/z ions

CID – collision-induced dissociation

takes place in ion trap in presenceof inert collision gas (He or Ar)

CID fragmentation of peptide YICDNQDTISSK

b ion series:

b1: Y-b2: YI-b3: YIC-b4: YICD-b5: YICDN-b6: YICDNQ-b7: YICDNQD-b8: YICDNQDT-b9: YICDNQDTI-b10: YICDNQDTIS-b11: YICDNQDTISS-

y ion series:

y1: K-y2: KS-y3: KSS-y4: KSSI-y5: KSSIT-y6: KSSITD-y7: KSSITDQ-y8: KSSITDQN-y9: KSSITDQND-y10: KSSITDQNDC-y11: KSSITDQNDCI-

y10+2H

y10+1

y2b2

y3

y4b3 y5b4y6

b5y7b6

y8

b7

y9

b8b9

y10

b10 y11b11

Y I C+57 D N Q D T I S S KK S S I T D Q N D C+57 I Y

m/z

Rel

ativ

e In

tens

ity

0%

25%

50%

75%

100%

0 250 500 750 1000 1250

1443.72 AMU, +2 H (Parent Error: 750 ppm)

+NH3-YICDNQDTISSK-COO-

Peptide sequence determinationby MS/MS fragmentation

902.4?

948.8?

952.4?

980.5?

1000.0?1008.5?1017.7?y7+2H

y11+2Hb6-H2O

y12+2H

b8-NH3y17-H2O-H2O+2H

b17-NH3-NH3+2Hy9+1

y10-H2O

y10+1

y11+1

y12+1

y14+1y15+1

b14-H2O

y3

b2

b3

y4

b4

y5b5

y6b6 y7

y8

b8y9

b9

y10

y11y12

b12

y13

b13

b14

b15

y16

C+57 C+57 A A D D K E A C+57 F A V E G P KK P G E V A F C+57 A E K D D A A C+57 C+57

m/z

Rel

ativ

e In

tens

ity

0%

25%

50%

75%

100%

0 250 500 750 1000 1250 1500 1750

1927.15 AMU, +2 H (Parent Error: 190 ppm)

• perform a theoretical trypsin digestion of all known proteins

• determine a theoretical MS/MS fragmentation for each peptide

• compare experimental spectrum to each theoretical spectrum

Peptide/protein identification by database searching

Mascot peptide scoring

Compilation of database search results

Challenges in mapping post-translational modifications

• Stoichiometry is often unfavorable

• Sequence coverage gaps

• Relatively unstable modifications are often lostduring peptide fragmentation

10.0 10.5 11.0 11.5 12.0 12.5 13.0 13.5 14.0 14.5 15.0Time (min)

0

10

20

30

40

50

60

70

80

90

100R

elat

ive

Abun

danc

e13.63

13.66

12.3111.85

11.25 14.2311.20 13.4012.6910.76 11.57 14.2812.7910.45

NL Bas FTM ms [37150 Cue

RLSVVGPPNR

chromatogram

phos

phor

ylat

ed p

eptid

e

unm

odifi

ed p

eptid

e

Protease digestion of proteins for MS analysis

Commonly used alternate proteases• AspN – cleaves on N-terminal side of Asp residues• GluC – cleaves on C-terminal side of Glu residues• LysC – cleaves on C-terminal side of Lys residues• ArgC – cleaves on C-terminal side of Arg residues

• chymotrypsin – cleaves on C-terminal side ofhydrophobic residues

• proteinase K – not sequence specific; used forlimited digestion

parent-98

b2 y2b3 y3 b4 b5y4 b7y6 y7 b8 y8b9 y9 b10 y10

K S E S+80 T S S Y N Y RR Y N Y S S T S+80 E S K

m/z

Rel

ativ

e In

tens

ity

0%

25%

50%

75%

100%

0 200 400 600 800 1000 1200 1400

1401.23 AMU, +2 H (Parent Error: 480 ppm)

“Typical” phosphopeptide spectrum:

+NH3-KSESTSSYNYR-COO-

neutral loss

Comparison ofETD and CIDfor phosphopeptideMS/MS

Kim and Pandey, Proteomics 12: 530-42 (2012)

Mapping of post-translational modifications requires

1) Identification of modified peptides

AND2) Localization of modified sites

PTM site localization using Ascore

Beausoleil et al., Nature Biotech. 24: 1285-92 (2006)

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