post-translational modification identification by mass ... ptm.pdfglu pro pro gln arg ala pro phe...
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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
Generating Peptides containing modified residue --Different Enzymes/Digestion Condition
Low population of modifications--Enrichment--Targeted Method (For better MSMS)
Difficulties for PTMs Identification
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
NL:5.98E5Base Peak m/z= 479.2634-479.2826 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:2.47E8Base Peak m/z= 85.0000-2000.0000 MS 17625_1_rerun
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)
Generating Peptides containing modified residue --Different Enzymes/Digestion Condition
Low population of modifications--Enrichment--Targeted Method
Modification not stable--Experiment Condition (reducing reagents, enzymes)--Fresh sample
Difficulties for PTMs Identification
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
Generating Peptides containing modified residue --Different Enzymes/Digestion Condition
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
Difficulties for PTMs Identification
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
Observed m/z =619.30162+
Theoretical m/z =619.29942+
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
Conservation of structure and mechanism by Trm5 enzymes RNA. 2013 Sep; 19(9): 1192–1199
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
Observed m/z =768.35733+
Theoretical m/z =768.35393+
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
Conservation of structure and mechanism by Trm5 enzymes RNA. 2013 Sep; 19(9): 1192–1199
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
Observed m/z =680.99663+
Theoretical m/z =680.99363+
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
Conservation of structure and mechanism by Trm5 enzymes RNA. 2013 Sep; 19(9): 1192–1199
“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