post-translational modification identification by mass ... ptm.pdfglu pro pro gln arg ala pro phe...

Post-translational Modification Identification by Mass Spectrometry

Liwen Zhang

Mass Spectrometry and Proteomics Facility

The Ohio State University

Summer Workshop 2016

PTM: Chemical Modifications on ProteinsIncreases the Functional Diversity of the ProteomeRegulate Activity, Localization and Interaction

Image from https://www.lifetechnologies.com/overview‐post‐translational‐modification.html

Lysine MethylationArginine Methylation

Acetylation

Phosphorylation

K‐GG

Ubiquinylation

K‐GGXXX

Sumoylation

Citrullination

Histone Modifications

Post-translational Modifications

ModificationModified Residue

Mass Shift (Da) Function

Disulphide bond formation

C ‐2.0157

protein stability

Methylation K/R 14.0157 regulation of gene expression

Hydroxylation P 15.9949 Protein stability and protein-ligand interaction

Oxidation of Met M 15.9949 usually introduced during digestion

Acetylation K 42.0106 Protein stability, protection of N terminus, regualtion of protein-DNA interactions

S‐Glutathionylation C 305.0682oxidative stress; preventing irreversible oxidation of protein thiol; control of cell-signaling pathways by modulating protein function

Phosphorylation S/T/Y 79.9663Signaling, activation/inactivation of enzyme activities, modulation of molecular interactions

Nitration Y 44.9851 oxidative damage during inflammation

Ubiquitination K 114.0429 destruction signal

Oxidation of Cys C 31.9721/47.9847 oxidative/nitrosative stress

Nitrosylation C 28.9902 oxidative/nitrosative stress

Glycosylation N/S/T‐‐‐‐‐‐‐‐

Excreted proteins, cell-cell recognition/signaling O-GlcNAc, reversible, regulatory functions

Method for PTM Detections

• Specific Fluorescence Dye: Pre-Q Diamond---PhosphorylationPre-Q Emerald---Glycosylation

• Specific Antibodies: Nitration; Phosphorylation; Methylation;Ubiquitination; Acetylation

• Mass Shift/PI Change: Gel Approaches

Pros: High sensitivity, Favor the modificationCons: Lack of accuracy

Interference from other residuesLack the ability to locate the actual modification sites

Mass Spectrometry!!!!!

Peptide Modifications Identification by MS/MS

• Virtually anything that shifts the mass can be determined by MS

• Using MS/MS allows for identification of the modification AND location

• You must have an idea of what modification you are looking for

• First use protein stain to examine various modifications

• Western analysis VS MS analysis

Cons: Detection based on ionization efficiency and abundance of the peptides, thus may not favor modified peptides.

Identification of PTMs:

Know the Modification (Stability, Mass Shift, possible modification site)

High Samples Purity

High Concentration

High Mass Accuracy/Resolution

300 400 500 600 700 800 900 1000 1100 1200 1300 1400m/z

1500

317.28y3

404.24y4

503.32y5

617.36y6

718.37y7

817.37y8

984.39y9

1083.45y10

1197.51y11

1311.59y12

1426.56y13

391.12b4

505.27b5

619.29b6

718.37b7

1484.46b14

984.39b9

1085.46b10

1199.51b11

1298.46b12

1385.54b13

886.36y9*

1099.56y11*

1213.70y12*

886.36b9*

1101.51b11*

1200.51b12*

1287.76b13*

1386.63b14*

1457.78b15*

Ser Val Asn Thr Val Val Asn Asn Asp

SerValAsnThr ValValAsnAsn

41T S S D N N V S(PO4)48 V T N V S V A K56

bn*=bn-H3PO4yn*=yn-H3PO4m/z=850.542+

802.432+

M-H3PO4

b13b13*b4 b6 b7 b15*

b11b11*b5

y9y9*

y10 y3y4y5y6y8 y7

b14b14*

y11y11*

y12y12*

y13

b10b12b12*

b9b9*

167.02 (87+80)Ser(PO4)

266.02 (87+80+99)Ser(PO4)-Val

Example 1 — Phosphorylation Identification by MS/MS

200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400m/z

241E A F P A Y246(N2O) R Q P P E S L253

b3 b4 b7 b8 b11

y3y4y8y9y10y11

219.15y2

348.10y3

445.22y4

542.31y5

670.43y6

826.20y7

1034.50y8

1105.51y9

1202.57y10

1349.69y11

675.552+

y11

348.10b3

445.22b4

1105.51b9

1202.57b10

880.42b7

1008.45b8

1331.59b11

601.992+

b10

666.502+

b11 709.872+

b12

Glu Pro Pro Gln Arg Ala Pro Phe

GluPro ProGlnPro

b9 b10 b12

y2y6y7 y5

Measured m/z = 775.612+

Theoreticalm/z = 775.372+

208.3 (163+45)Tyr(N2O)

Example 2 — Nitration Identification by MS/MS

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

460.24y4

623.30y5

736.40y6

823.41y7

910.39y8

1023.55y9

1151.56y10

1280.54y11

1431.51y12

1488.48y13

1617.48y14

1745.66y15

1858.62y16

930.162+

y16

1014.182+

b19

979.482+

y17

584.26b6

713.38b7

770.40b8

921.28b9

1050.21b10

1178.34b11

1291.39b12

1378.34b13

1465.42b14

1578.48b15

1741.58b16

1798.75b17

1913.59b18

873.542+

y15

E G E K I S S L Y G D

EGEKISSLY Q L

529V G S V L Q E G C(SO3) E K I S S L Y G D L R548y4y5y7y8y9y10y11y12y14

b6 b7 b8 b9 b10 b11 b12 b13 b14 b15 b16

y6y13y15y17 y16

b17 b18 b19

150.88(103+48)C(SO3)

150.97(103+48)C(SO3)

Example 3 — Identification of Cysteine Oxidation by MS/MS

500 600 700 800 900 1000 1100 1200 1300 1400 1500

m/z

638.892+

b8

638.312+

y9 1274.57y9

738.122+

b10

794.642+

b11

795.572+

y11

960.47y7

573.11b4

759.32b5

906.56b6

1062.49b7

1276.67b8

1474.67b10

186.21Trp

97.24Phe

155.93Arg

198.00 (97+101)Pro-Thr

900E E W K W F R C907(DMPO) P T L L911

b4 b5 b6 b7 b8 b10 b11

y11 y9 y7

Theoretical m/z=859.942+

Observed m/z=859.892+

DMPO=C6H9NO (111.0684 Da)

214.18(103+111)C-DMPO

Example 4 — Identification of DMPO Adduct by MS/MS

200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600m/z

712.632+

y14

753.582+

b14

276.20y2

347.18y3

446.27y4

547.27y5

618.41y6

717.41y7 774.38

y8

911.36y9 1109.47

y11

1166.53y12

1295.49y13

1423.55y14

1010.47y10

357.19b2

486.30b3

543.30b4

642.31b5

741.36b6

878.49b7

935.48b8

1034.45b9

1206.44b11

1305.59b12

1376.51b13

1505.59b14

1105.51b10

44X K E G V V H G V A T V A E K60y4 y2

b13 b14

y5

b12

y6

b10 b11

y8y10 y9

b7 b9

y11y12

b4 b5

y13

b3 b6 b8

y7

b2

y3y14

70.98A

99.09V

101.00T

71.13A

99.00V

56.97G

136.98H

99.11V

99.00V

57.06G

128.96E

128.06K

70.92A

99.15V

100.93T

71.06A

97.97V

56.99G

137.13H

99.05V

99.01V

57.00G

129.11E

129.98E

Mass of X=(M+H)-y14=826.45*2-1-1423.55=228.35

Mass of X=b2-Mass of Lysine-H=357.19-128.02-1.01=228.16

PTM Identification: when modification is on the terminus of the peptide

44X K E G V V H G V A T V A E K60

Theoretical m/z 826.45432+

Observed m/z 826.45732+

Mass Error 3.63 ppm

y4 y2

b13 b14

y5

b12

y6

b10 b11

y8y10 y9

b7 b9

y11y12

b4 b5

y13

b3 b6 b8

y7

b2

y3y14

Modification occurs on lysine residueMass of modification =Mass of X-Mass of Lysine=228.16-128.09=100.05DaSuccinylation: Addition of –COCH2CH2CO- to lysine residue (+100.0115Da)

High mass accuracy instrument neededUse high mass accuracy for MS/MS tooMSn can be performed to resolve the structure of modification if necessary

PTM Identification: when modification is on the terminus of the peptide

Generating Peptides containing modified residue --Different Enzymes/Digestion Condition

Low population of modifications (usually less than 1%)--Enrichment--Targeted Method

Modification not stable --Experiment Condition (reducing reagents, enzymes)--Fresh sample

Modification labile in MS analysis--Neutral Loss Method--Different Fragmentation Method

Difficulties for PTMs Identification

Trypsin K-X and R-X

Endoprotease Lys-C K-X except when X = P

Endoprotease Arg-C R-X except when X = P

Endoprotease Asp-N X-D

Endoprotease Glu-C E-X except when X = P

Chymotrypsin L-X, F-X, Y-X and W-X

Cyanogen Bromide X-M

Enzymes for Proteome Research

1 MAALKLLSSG LRLGASARSS RGALHKGCVC YFSVSTRHHT KFYTDPVEAV 51 KDIPNGATLL VGGFGLCGIP ENLIGALLKT GVKDLTAVSN NAGVDNFGLG

101 LLLRSKQIKR MISSYVGENA EFERQFLSGE LEVELTPQGT LAERIRAGGA 151 GVPAFYTSTG YGTLVQEGGS PIKYNKDGSV AIASKPREVR EFNGQHFILE 201 EAITGDFALV KAWKADRAGN VIFRKSARNF NLPMCKAAGT TVVEVEEIVD 251 IGSFAPEDIH IPKIYVHRLI KGEKYEKRIE RLSLRKEGDG KGKSGKPGGD 301 VRERIIKRAA LEFEDGMYAN LGIGIPLLAS NFISPNMTVH LQSENGVLGL 351 GPYPLKDEAD ADLINAGKET VTVLPGASFF SSDESFAMIR GGHVNLTMLG 401 AMQVSKYGDL ANWMIPGKMV KGMGGAMDLV SSSKTKVVVT MEHSAKGNAH 451 KIMEKCTLPL TGKQCVNRII TEKGVFDVDK KNGLTLIELW EGLTVDDIKK 501 STGCDFAVSP NLMPMQQIST

Tryspin: 309AALEFEDGMYANLGIGIPLLASNFISPNMTVHLQSENGVLGLGPYPLK356

Chymotrypsin: 329ASNF332, GluC: 315DGMYANLGIGIPLLASNFISPNMTVHLQSE344

Phosphorylation

Using the CORRECT Enzyme

Optimize Digestion Conditions1 MADESSDAAG EPQPAPAPVR RRSSANYRAY ATEPHAKKKS KISASRKLQL

51 KTLMLQIAKQ EMEREAEER R GEKGRVLRTR CQPLELDGLG FEELQDLCRQ101 LHARVDKVDE ERYDVEAKVT KNITEIADLT QKIYDLRGKF KRPTLRRVRI151 SADAMMQALL GTRAKESLDL RAHLKQVKKE DIEKENREVG DWRKNIDALS201 GMEGRKKKFE G

Sequence coverage: 97.2%

Start Observed Mr(expt) Mr(calc)

ppm M Score Peptide

AA2-20 932.9369 1863.8592 1863.9 -0.34 0 104 M.ADESSDAAGEPQPAPAPVR.RAA2-21 674.3265 2019.9576 2020 -1.67 1 95 M.ADESSDAAGEPQPAPAPVRR.RAA2-22 726.3605 2176.0598 2176.1 -1.06 2 66 M.ADESSDAAGEPQPAPAPVRRR.SAA23-37 555.9385 1664.7936 1664.8 1.78 1 55 R.SSANYRAYATEPHAK.KAA23-39 641.3364 1920.9874 1921 3.59 3 44 R.SSANYRAYATEPHAKKK.SAA29-37 494.2502 986.4858 986.48 3.69 0 26 R.AYATEPHAK.KAA29-39 622.3464 1242.6782 1242.7 4.97 2 44 R.AYATEPHAKKK.SAA29-41 486.9404 1457.7994 1457.8 0.25 3 35 R.AYATEPHAKKKSK.IAA29-46 658.37 1972.0882 1972.1 1.43 4 34 R.AYATEPHAKKKSKISASR.KAA47-59 382.7495 1526.9691 1527 7.14 2 35 R.KLQLKTLMLQIAK.QAA52-64 530.952 1589.8341 1589.8 4.51 1 85 K.TLMLQIAKQEMER.EAA52-69 735.7083 2204.1029 2204.1 4.57 2 49 K.TLMLQIAKQEMEREAEER.RAA60-69 436.1964 1305.5675 1305.6 4.31 1 25 K.QEMEREAEER.RAA76-99 730.1263 2916.476 2916.5 5.98 2 31 R.VLRTRCQPLELDGLGFEELQDLCR.QAA79-99 850.4134 2548.2183 2548.2 5.24 1 95 R.TRCQPLELDGLGFEELQDLCR.QAA81-99 1146.5381 2291.0616 2291.1 2.36 0 115 R.CQPLELDGLGFEELQDLCR.QAA81-104 725.1068 2896.3981 2896.4 0.75 1 69 R.CQPLELDGLGFEELQDLCRQLHAR.VAA105-118 565.6145 1693.8217 1693.8 3.43 2 41 R.VDKVDEERYDVEAK.VAA105-121 1012.028 2022.0415 2022 7.19 3 39 R.VDKVDEERYDVEAKVTK.NAA119-132 787.4489 1572.8833 1572.9 7 1 63 K.VTKNITEIADLTQK.IAA119-137 745.4208 2233.2405 2233.2 3.91 2 65 K.VTKNITEIADLTQKIYDLR.GAA119-139 605.5953 2418.3522 2418.3 1.68 3 54 K.VTKNITEIADLTQKIYDLRGK.FAA122-132 623.3372 1244.6599 1244.7 -1.06 0 79 K.NITEIADLTQK.IAA122-137 953.5199 1905.0252 1905 2.38 1 66 K.NITEIADLTQKIYDLR.GAA122-139 697.7211 2090.1414 2090.1 2.03 2 48 K.NITEIADLTQKIYDLRGK.FAA138-146 368.2345 1101.6816 1101.7 4.08 2 26 R.GKFKRPTLR.RAA138-147 420.2665 1257.7776 1257.8 -0.46 3 26 R.GKFKRPTLRR.VAA148-163 866.964 1731.9134 1731.9 0.61 1 79 R.VRISADAMMQALLGTR.AAA150-163 739.3765 1476.7385 1476.7 -2.94 0 107 R.ISADAMMQALLGTR.AAA164-171 466.2661 930.5176 930.51 4.52 1 58 R.AKESLDLR.A

AA176-187 505.9445 1514.8118 1514.8 4.36 3 59 K.QVKKEDIEKENR.E

AA188-205 678.3385 2031.9937 2032 6.93 2 43 R.EVGDWRKNIDALSGMEGR.KAA194-205 645.8306 1289.6466 1289.6 5.29 1 78 R.KNIDALSGMEGR.KAA195-205 581.7822 1161.5499 1161.5 4.39 0 95 K.NIDALSGMEGR.KAA195-206 645.8309 1289.6472 1289.6 5.77 1 54 K.NIDALSGMEGRK.KAA195-207 709.878 1417.7414 1417.7 4.74 2 46 K.NIDALSGMEGRKK.KAA195-211 627.329 1878.9651 1879 1.58 4 29 K.NIDALSGMEGRKKKFEG.-

Trypsin; 1:200; RT; 20 min Digestion

Adding Ion Pairing Reagent

AA Start - End Observed Mr(expt) Mr(calc) ppm M Score Peptide

22-28 467.2008 932.387 932.387 0.53 1 34 R.RSSANYR.A + Phospho (ST)

23-28 349.1678 696.321 696.319 2.7 0 42 R.SSANYR.A

23-28 349.1684 696.3222 696.319 4.54 0 43 R.SSANYR.A

1 MADESSDAAG EPQPAPAPVR RRSSANYRAY ATEPHAKKKS KISASRKLQL 51 KTLMLQIAKQ EMEREAEERR GEKGRVLRTR CQPLELDGLG FEELQDLCRQ

101 LHARVDKVDE ERYDVEAKVT KNITEIADLT QKIYDLRGKF KRPTLRRVRI 151 SADAMMQALL GTRAKESLDL RAHLKQVKKE DIEKENREVG DWRKNIDALS 201 GMEGRKKKFE G

MSMS for RSS(PO4)ANYR MSMS for SSANYR


Low population of modifications--Enrichment--Targeted Method (For better MSMS)


PTM Enrichment Techniques (Start with mg of samples)

Acetylation: Anti-acetyl lysine polyclonal antibody

Phosphopeptides: Immobilized Metal Affinity Chromatography (Fe3+)Metal Oxide Affinity Chromatography (TiO2, ZrO2)Reversible Covalent Binding

(Techniques for phosphopeptide enrichment prior to analysis by mass spectrometry. Mass Spectrom Rev. 2010 Jan-Feb;29(1):29-54.)

Phospho-Threonine Antibody (P-Thr-Polyclonal)

Nitration: Anti‐Nitrotyrosine polyclonal antibody

Ubiquitination: K-ε-GG–specific antibody (enrichment has to be done prior to other treatment on lysine)

Glycopeptides: Lectin affinity enrichmentCovalent InteractionsChromatographic separation

(Glycopeptide enrichment and separation for protein glycosylation analysis., J Sep Sci. 2012 Sep;35(18):2341-72).

Methylation: Anti-methyl lysine/araginine antibody

TiO2 beads

extract cells

protein

In-solution digestion

mLKB1

cell lysis

peptides

Slide courtesy of Nilini S. Ranbaduge

Enrichment for Phosphorylation

Phosphopeptides/proteins Identification

Slide courtesy of Nilini S. Ranbaduge

SIC of 516.77932+ (SLVLC(CAM)TPSR)

SIC of 479.27302+ (SLVLfGlyTPSR )

100:1

RT: 19.61 - 39.93

20 22 24 26 28 30 32 34 36 38Time (min)

0

10

20

30

40

50

60

70

80

90

1000

10

20

30

40

50

60

70

80

90

100

Relative Abundance

27.80

27.9327.74

27.69 27.96

28.01

27.6128.06

27.55

27.5028.13

27.4428.21

27.3828.3627.3028.56

27.19 29.0725.9520.90 30.13 31.4825.54 33.67 36.0434.17 38.6538.1521.45 23.8222.8927.80

27.82

27.74

27.66

27.96

27.61

28.0127.55

28.0627.5039.60

35.9128.1336.0927.44 39.55

27.3628.21 31.90 32.0927.25 39.4936.2531.7628.64

27.06 38.6833.4125.8521.30 35.5921.7520.02 23.65 33.8624.59

NL:4.05E7Base Peak m/z= 516.7690-516.7896 MS 17625_1_rerun


MS/MS of 516.77932+ (SLVLC(CAM)TPSR) MS/MS of 479.27302+ (SLVLfGlyTPSR )

NOT Observed!!!!

Full Scan

17625_1_rerun #2715-9589 RT: 18.73-51.92 AV: 99 NL: 5.71E3T: Average spectrum MS2 516.79 (2715-9589)

150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e Abu

ndanc

e

733.45

620.39

507.76

832.54

462.29

416.76

300.06413.05

201.05 487.72

544.28 575.37359.31173.20

674.26255.07

704.44367.27 423.26347.30 407.78 602.00

288.20 886.49656.23319.31 804.48213.06 773.42

Val Leu Cys(CAM) Thr

Thr

Cys(CAM) ValLeu

Example---Targeted MS Scan (for Known Modifications)

RT: 19.94 - 50.13

20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50Time (min)

0

10

20

30

40

50

60

70

80

90

1000

10

20

30

40

50

60

70

80

90

100

Relative Abundance

35.88

42.9541.90

49.2343.66

49.0744.6735.99

40.05

40.1536.06

40.20 45.5136.2728.36

28.2128.13

28.54 34.20 36.46 38.2646.3933.80 46.58

32.0428.6728.08 32.09 36.5747.1631.93

24.09 27.55 47.4728.9023.8521.16 29.16

41.95

42.09 44.90

35.9545.16

44.8141.8635.8240.13 43.30 49.38

28.69 46.0039.9536.39 40.32 48.1234.2528.92 36.5932.4428.26 38.2132.1425.8224.7020.00 22.08


NL:8.17E8Base Peak MS 17625_1_Target_2

Full Scan

Targeted @ 479.27302+(SLVLfGlyTPSR )/516.77932+(SLVLC(CAM)TPSR)

MS/MS of 516.77932+ (SLVLC(CAM)TPSR)MS/MS of 479.27302+ (SLVLfGlyTPSR )

Val Leu Cys(CAM) Thr

Thr

Cys(CAM) ValLeu

17625_1_Target_2 #667-2166 RT: 19.74-39.25 AV: 264 NL: 3.92E4T: ITMS + c ESI d Full ms2 [email protected] [130.00-1045.00]

150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rela

tive A

bun

dance

733.44

620.39

832.51

507.74

416.77

460.36

300.06

413.10201.05

359.31173.20 573.18

674.27255.09

407.74555.09 656.23 715.43

602.35213.08 498.76 814.46545.24 858.31310.81262.26 430.13 771.31689.49 886.44

Pro Ser

17625_1_Target_2 # 799-1295 RT: 22.03-28.48 AV: 40 NL: 1.03E3T: ITMS + c ESI d Full ms2 [email protected] [120.00-970.00]

150 200 250 300 350 400 450 500 550 600 650 700 750 800m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lativ

e A

bund

anc

e

581.28

359.31

470.22

460.28

757.45545.38 658.34

413.05

402.85328.66299.98

631.30201.02 446.40594.32173.19

388.76 517.26255.10 671.51730.39229.03 781.32702.58

Val LeufGly Thr

ValLeufGlyThr

Example---Targeted MS Scan (for Known Modifications)


Low population of modifications--Enrichment--Targeted Method

Modification not stable--Experiment Condition (reducing reagents, enzymes)--Fresh sample


m/z400 600 800 1000 1200 1400 1600250

Chain A

Chain B

185N A C187 G S G Y D F D V F V V R199

138L V E G C142 L V G G R146

S

S

408.17 465.20 552.31 609.30 772.30 1034.35 1149.39 1248.41 1395.53 1494.50 1593.59

289.23y3B

342.33b3B

501.10y5B

274.25y2A

373.31y3A

520.42y4A

619.48y5A

734.51y6A

1303.64y11A

1159.62y9A

1216.57y10A

1360.66y12A

996.57y8A

y2Ay3Ay4Ay5Ay6Ay8Ay10Ay11Ay12A y9A

V F V D FD Y G S G

VFVDFDYGSG V

y3By5B

b3B

A

B

Adjust Experiment Condition to Preserve Modifications

Nitrosylation and Biotin Switch

http://www.pnas.org/content/103/19/7420/F1.expansion

200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400m/z

194A G R P G M G V O202 G P E T S L208

y4y5y7y8y9y10y12

b3 b5 b6 b7 b8 b9 b10 b11 b12 b13 b14

y6 y3

Theoretical m/z =783.40892+, Observed m/z =783.40802+, Mass Error =1.15ppm

674.472+

b13

285.21b3

439.23b5

570.30b6

627.43b7

1020.49b10

726.35b8

963.39b9

1117.55b11

1347.71b13

1246.54b12

718.062+

b14

623.822+

b12

154.13 (97+57)P‐G

130.97M

57.13G

98.92V

237.04O

101.17T

57.10G

97.06P

128.99E

320.13y3

449.05y4

546.24y5

603.21y6

840.38y7

939.24y8

996.30y9

1127.37y10

1281.45y12

128.92E

97.19P

56.97G

237.17 O

98.86V

57.06G

131.07M

154.08 (57+97)G‐P

O=Pyrrolysine

Using Different Enzyme to Protect Modifications

MQIFVKTLTG KTITLEVEPS DTIENVKAKI QDKEGIPPDQ QRLIFAGKQL EDGRTLSDYN IQKESTLHLV LRLRGG

Unmodified

+1Ub

+2Ub

+3Ub

Trypsin!!!

Choose a Good Enzyme to Generate Signature Modification Group—the Identification of Ubiquitination by MS/MS

A proteomics approach to understanding protein ubiquitination Nature Biotechnology 21, 921 - 926 (2003)

Choose a Good Enzyme to Generate Signature Modification Group—the Identification of Ubiquitination by MS/MS

LTQ DATA—Fresh Sample

Sequence Coverage 39%; T95 was phosphorylatd

1 MSDKSELKAE LERKKQRLAQ IREEKKRKEE ERKKKETDQK KEAVAPVQEE

51 SDLEKKRREA EALLQSMGLT PESPIVFSEY WVPPPMSPSS KSVSTPSEAG

101 SQDSGDGAVG SRTLHWDTDP SVLQLHSDSD LGRGPIKLGM AKITQVDFPP

151 REIVTYTKET QTPVMAQPKE DEEEDDDVVA PKPPIEPEEE KTLKKDEEND201 SKAPPHELTE EEKQQILHSE EFLSFFDHST RIVERALSEQ INIFFDYSGR

251 DLEDKEGEIQ AGAKLSLNRQ FFDERWSKHR VVSCLDWSSQ YPELLVASYN

301 NNEDAPHEPD GVALVWNMKY KKTTPEYVFH CQSAVMSATF AKFHPNLVVG351 GTYSGQIVLW DNRSNKRTPV QRTPLSAAAH THPVYCVNVV GTQNAHNLIS401 ISTDGKICSW SLDMLSHPQD SMELVHKQSK AVAVTSMSFP VGDVNNFVVG

451 SEEGSVYTAC RHGSKAGISE MFEGHQGPIT GIHCHAAVGA VDFSHLFVTS501 SFDWTVKLWT TKNNKPLYSF EDNADYVYDV MWSPTHPALF ACVDGMGRLD

551 LWNLNNDTEV PTASISVEGN PALNRVRWTH SGREIAVGDS EGQIVIYDVG

601 EQIAVPRNDE WARFGRTLAE INANRADAEE EAATRIPA

Detected 30 times!!!

Orbitrap XL Data‐‐‐Two Days old, Stored at 4C

Sequence Coverage 53%; No Phosphorylation Detected

1 MSDKSELKAE LERKKQRLAQ IREEKKRKEE ERKKKETDQK KEAVAPVQEE

51 SDLEKKRREA EALLQSMGLT PESPIVFSEY WVPPPMSPSS KSVSTPSEAG

101 SQDSGDGAVG SRTLHWDTDP SVLQLHSDSD LGRGPIKLGM AKITQVDFPP

151 REIVTYTKET QTPVMAQPKE DEEEDDDVVA PKPPIEPEEE KTLKKDEEND

201 SKAPPHELTE EEKQQILHSE EFLSFFDHST RIVERALSEQ INIFFDYSGR

251 DLEDKEGEIQ AGAKLSLNRQ FFDERWSKHR VVSCLDWSSQ YPELLVASYN301 NNEDAPHEPD GVALVWNMKY KKTTPEYVFH CQSAVMSATF AKFHPNLVVG

351 GTYSGQIVLW DNRSNKRTPV QRTPLSAAAH THPVYCVNVV GTQNAHNLIS

401 ISTDGKICSW SLDMLSHPQD SMELVHKQSK AVAVTSMSFP VGDVNNFVVG451 SEEGSVYTAC RHGSKAGISE MFEGHQGPIT GIHCHAAVGA VDFSHLFVTS501 SFDWTVKLWT TKNNKPLYSF EDNADYVYDV MWSPTHPALF ACVDGMGRLD

551 LWNLNNDTEV PTASISVEGN PALNRVRWTH SGREIAVGDS EGQIVIYDVG

601 EQIAVPRNDE WARFGRTLAE INANRADAEE EAATRIPA


Low population of modifications--Enrichment--Targeted Method

Modification not stable--Experiment Condition (reducing reagents, enzymes)--Fresh sample

Modification labile in MS analysis--Neutral Loss Method--Different Fragmentation Method


Scan ScanDissociate

Neutral Loss Scan

http://www.biotechniques.com/BiotechniquesJournal/2006/June/Analysis-of-posttranslational-modifications-of-proteins-by-tandem-mass-spectrometry

Hydrothermal synthesis of α-Fe2O3@SnO2 core–shell nanotubes for highly selective enrichment of phosphopeptidesfor mass spectrometry analysis Nanoscale, 2010,2, 1892-1900

391.23b3

504.24y4

1070.62b9 1561.73

y13 1777.84b15

391.23b3

292.16b2

883.43y8*

784.36y7*

996.51y9* 1125.55

y10*

1070.62b9

1238.64y11*

1463.75y13*

1562.82y14*

YKVPQLEIVPNsAEER

Alpha Casein

• Help Identify the Sequencebut don’t Give Informationabout the Location of theModification if Multiple SitesCoexisting.

• Sensitive not Ideal

NL of Phosphorylation:98 (+1), 49 (+2)…

ECD/ETD: Keep the Phosphorylation Intact!!!

Electron Capture Dissociation (ECD, ICR)/ Electron Transfer Dissociation (ETD,Traps) will Generate Peptide Fragments without Losing the SidechainPhosphorylation

Sweet, Anal. Chem. 2006, 78, 7563-7569

H2N C C

O

N

H

R1

H

C C

O

N

H

R2

H

C C

O

N

H

Rn-1

H

C C

ORn

OH…

b1 a2 b2 c2 an-1 bn-1 cn-1

x1 y1 z1xn-2 yn-2 zn-2xn-1 yn-1

c1

zn-1

a1

CID: a, b and y ion, loss of H2O, NH3, side-chain ECD/ETD: c, y and z ions

H3PO4 Group may Leave upon CID Dissociation; Actual Amino AcidLocation of H3PO4 Group May not be Clear if >2 Sites in the Sequence

Leann M. Mikesh et al. Biochimica et Biophysica Acta 1764 (2006) 1811–1822

FS(PO4)ADYHSHLDSLSKPSEYSDLK

200 300 400 500 600 700 800 900 1000 1100 1200 1300

m/z

CID

ETD

244.15z2

332.07c3

359.20z3

403.18c3

446.25z4

518.17c4

609.31z5

681.24c5

738.34z6

818.37c6

825.38z7

905.32c7

1042.45c8

1137.532+

z20

1222.032+

z21

1103.022+

z19963.972+

z17

1239.742+

c211182.492+

c20

1081.732+

c18

999.862+

c17

652.664+

MH4+

869.404+

MH3+

837.113+

MH‐H3PO43+

KESPPT(PO4)PDQGASSHGPGHAEENGFITFSQYSSESDTTADYTTEK

CID

ETD

Unknown Truncation Sites Identification

Separate Suspected Truncation Product(s) from Intact Protein

Intact MS Measurement

Top down Sequencing

Separation on 1D SDS PAGE

Cut the band (truncated protein)Digestion LC/MSMS

Protein sequence

MASCOT

Suggested truncation sites

Peptide Sequencing

Search against new sequences

Validation

Conservation of structure and mechanism by Trm5 enzymes RNA 2013 Sep; 19(9): 1192–1199

MASCOT RESULT: 1 MVLWILWRPF GFSGRFLKLE SHSITESKSL IPVAWTSLTQ MLLEAPGIFL 51 LGQRKRFSTM PETETHERET ELFSPPSDVR GMTKLDRTAF KKTVNIPVLK 101 VRKEIVSKLM RSLKRAALQR PGIRRVIEDP EDKESRLIML DPYKIFTHDS 151 FEKAELSVLE QLNVSPQISK YNLELTYEHF KSEEILRAVL PEGQDVTSGF 201 SRIGHIAHLN LRDHQLPFKH LIGQVMIDKN PGITSAVNKI NNIDNMYRNF 251 QMEVLSGEQN MMTKVRENNY TYEFDFSKVY WNPRLSTEHS RITELLKPGD 301 VLFDVFAGVG PFAIPVAKKN CTVFANDLNP ESHKWLLYNC KLNKVDQKVK 351 VFNLDGKDFL QGPVKEELMQ LLGLSKERKP SVHVVMNLPA KAIEFLSAFK 401 WLLDGQPCSS EFLPIVHCYS FSKDANPAED VRQRAGAVLG ISLEACSSVH 451 LVRNVAPNKE MLCITFQIPA SVLYKNQTRN PENHEDPPLK RQRTAEAFSD 501 EKTQIVSNT

~260AA =~27‐28kDa

Example---Unknown Truncation Sites Identification

Constructed new sequences (a total of 24) and searched the data against these sequences:>16502 Sequence 1 (241-509)NNIDNMYRNFQMEVLSGEQNMMTKVRENNYTYEFDFSKVYWNPRLSTEHSRITELLKPGDVLFDVFAGVGPFAIPVAKKNCTVFANDLNPESHKWLLYNCKLNKVDQKVKVFNLDGKDFLQGPVKEELMQLLGLSKERKPSVHVVMNLPAKAIEFLSAFKWLLDGQPCSSEFLPIVHCYSFSKDANPAEDVRQRAGAVLGISLEACSSVHLVRNVAPNKEMLCITFQIPASVLYKNQTRNPENHEDPPLKRQRTAEAFSDEKTQIVSNT <>16502 Sequence 2 (242-509)NIDNMYRNFQMEVLSGEQNMMTKVRENNYTYEFDFSKVYWNPRLSTEHSRITELLKPGDVLFDVFAGVGPFAIPVAKKNCTVFANDLNPESHKWLLYNCKLNKVDQKVKVFNLDGKDFLQGPVKEELMQLLGLSKERKPSVHVVMNLPAKAIEFLSAFKWLLDGQPCSSEFLPIVHCYSFSKDANPAEDVRQRAGAVLGISLEACSSVHLVRNVAPNKEMLCITFQIPASVLYKNQTRNPENHEDPPLKRQRTAEAFSDEKTQIVSNT <…..

>16502 Sequence 23 (263-509)TKVRENNYTYEFDFSKVYWNPRLSTEHSRITELLKPGDVLFDVFAGVGPFAIPVAKKNCTVFANDLNPESHKWLLYNCKLNKVDQKVKVFNLDGKDFLQGPVKEELMQLLGLSKERKPSVHVVMNLPAKAIEFLSAFKWLLDGQPCSSEFLPIVHCYSFSKDANPAEDVRQRAGAVLGISLEACSSVHLVRNVAPNKEMLCITFQIPASVLYKNQTRNPENHEDPPLKRQRTAEAFSDEKTQIVSNT <>16502 Sequence 24 (264-509)KVRENNYTYEFDFSKVYWNPRLSTEHSRITELLKPGDVLFDVFAGVGPFAIPVAKKNCTVFANDLNPESHKWLLYNCKLNKVDQKVKVFNLDGKDFLQGPVKEELMQLLGLSKERKPSVHVVMNLPAKAIEFLSAFKWLLDGQPCSSEFLPIVHCYSFSKDANPAEDVRQRAGAVLGISLEACSSVHLVRNVAPNKEMLCITFQIPASVLYKNQTRNPENHEDPPLKRQRTAEAFSDEKTQIVSNT <

MASSMATRIX Results:hit# score decoy% protein descriptionhit1 3533 0.00% 16502 Sequence 12 (252-509)hit2 3522 0.00% 16502 Sequence 23 (263-509)hit3 3519 0.00% 16502 Sequence 14 (254-509)hit4 3512 0.00% 16502 Sequence 21 (261-509)

Example---Unknown Truncation Sites Identification

252M E V L S G E Q N M M T K264

Observed m/z =749.34412+

Theoretical m/z =749.34092+

Mass Error=4.27ppm

b8b5 b9b6b2 b3

y9 y8 y5 y4 y3 y2

b10

y12 y11 y10

200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400

m/z

400

800

1200

1400

Inte

nsi

ty

1200

617.17b6

874.34b8

1119.23b10

260.92b2

360.13b3

473.13b4

560.08b5

248.21y2

379.20y3

510.34y4

624.17y5

1366.52y12

1237.34y11

938.20y8

1138.37y10

99.21Val

113.00Leu

131.03Met

130.98Met

101.05Thr

131.14Met

130.99Met

113.83Asn

314.03 (57+129+128)Gly-Glu-Gln

87.07Ser

113.10Leu

98.97Val

129.18Leu

1025.27y9

988.20b9

1250.21b11

1351.26b12

113.86Asn

257.17(128+129)Glu-Gln

57.09Gly

86.95Ser

b4 b11 b12

Conservation of structure and mechanism by Trm5 enzymes RNA. 2013 Sep; 19(9): 1192–1199

254V L S G E Q N M M T K264

200 300 400 500 600 700 800 900 1000 1100 1200

m/z

1000

2000

3000

4000

5000

Inte

nsi

ty

859.15b8

990.43b9

1091.04b10

213.01b2

300.13b3

486.24b5

728.26b7

248.11y2

379.17y3

510.11y4

624.08y5

752.17y6

881.22y7

938.30y8

1138.25y10

1025.23y9

610.232+

M‐H2O



Mass Error=3.55ppm

b8b5 b9b7b2 b3

y7 y6 y5 y4 y3 y2

b10

y10 y9 y8

87.12Ser

186.11 (57+129)Gly-Glu

242.02 (128+114)Gln-Asn

130.89Met

131.28Met

100.61Thr

130.94Met

131.06Met

113.97Asn

128.09Gln

129.05Glu

57.08Gly

86.93Ser

113.02Leu


261M M T K V R E N N Y T Y E F D F S K278

200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600

m/z

250

500

750

1000

1250

1350

Inte

nsi

ty

234.08y2

381.14y3

496.40y4

643.26y5

772.16y6

935.14y7

1036.28y8

1199.31y9

1313.47y10

1427.34y11

1556.88y12

856.742+

y13

906.412+

y14906.412+

y15

1020.812+

y161086.462+

y17

263.06b2

364.12b3

492.11b4

747.66b6

438.922+

b7552.892+

b9684.622+

b11

830.872+

b13

904.472+

b14

961.622+

b15

1078.652+

b17634.302+

b10

b14b7 b9b6b2 b3 b10b4 b11 b13 b17b15

y7 y6 y5 y4 y3 y2y10 y9 y8y14 y13 y12 y11y17 y16 y15



Mass Error=4.42ppm

147.06Phe

115.26Asp

146.86Phe

128.90Glu

162.98Tyr

101.14Thr

163.03Tyr

114.16Asn

113.87Asn

129.54Glu

99.34Val

127.78Lys

101.02Thr

131.30Met

101.06Thr

127.99Lys

255.55 (99+156)Val-Arg


300 400 500 600 700 800 900 1000 1100 1200 1300 1400

m/z

263T K V R E N N Y T Y E F D F S K278

b14b7 b9b6 b8b3 b10b4 b11 b13 b15

y7 y6 y5 y4 y3y10 y9 y8y14 y13 y12



Mass Error=4.40ppm

200

400

600

800

900

Inte

nsi

ty

381.32y3

496.36y4

643.34y5

772.20y6

935.40y7

1036.27y8

1199.22y9

1313.39y10

778.642+

y12

906.362+

y14

115.04Asp

146.98Phe

128.86Glu

163.20Tyr

100.87Thr

162.95Tyr

114.17Asn

156.60Arg

856.942+

y13

485.56b4

728.46b6

842.88b7

1005.38b8

1106.55b9

1269.44b10

830.892+

b13

948.152+

b15

699.792+

b11

904.692+

b14307.442+

b5

98.84Val

242.90 (129+114)Glu-Asn

114.42Asn

162.50Tyr

101.17Thr

162.89Tyr


“The cell surface landscape is richlydecorated with oligosaccharides anchoredto proteins or lipids within the plasmamembrane. Cell surface oligosaccharidesmediate the interactions of cells with eachother and with extracellular matrixcomponents.” Science 291:2337

Why Glycosylation Investigation is SO HARD???

• Not only Protein Modification Requiring Detailed Structural Characterization(sugar chain/branched)

• Poor Ionization efficiency (large mass increase, sometimes has negative charge,hydrophobicity..)

• Heterogeneity• Large Size

Glycosylation—A Different Story

Determination of site-specific glycan heterogeneity on glycoproteins Nature Protocols 7, 1285–1298 (2012)

• Enrichment (Lectin Affinity Binding)

• Derivatization (Permethylation)

• LC Separation (PGC Column for Glycans, HILIC Column for Glycopeptides)

• Both MALDI and ESI will Work• High Mass Accuracy Desired• Negative Mode Works Better• ETD Improves the MSMS Identification

post-translational modification identification by mass ... ptm.pdfglu pro pro gln arg ala pro phe...

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