post-translational modification identification by mass spectrometry · 2019. 12. 18. ·...
TRANSCRIPT
Post-translational Modification Identification by Mass Spectrometry
Liwen ZhangMass Spectrometry and Proteomics Facility
The Ohio State University
Summer Workshop 2018
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
Post-translational Modifications
Modification Modified Residue Mass Shift (Da) Function
Disulphide bond formation C -2.0157
protein stabilityMethylation K/R 14.0157 regulation of gene expressionHydroxylation P 15.9949 Protein stability and protein-ligand interactionOxidation of Met M 15.9949 usually introduced during digestionAcetylation K 42.0106 Protein stability, protection of N terminus, regualtion of protein-DNA interactions
S-Glutathionylation C 305.0682 oxidative stress; preventing irreversible oxidation of protein thiol; control of cell-signaling pathways by modulating protein function
Phosphorylation S/T/Y 79.9663 Signaling, activation/inactivation of enzyme activities, modulation of molecular interactions
Nitration Y 44.9851 oxidative damage during inflammation
Ubiquitination K 114.0429 destruction signalOxidation 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
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 by MS:
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
167.02 (87+80)Ser(PO4)
Val Asn Asn Asp
SerValAsnThr Val
266.02 (87+80+99)Ser(PO4)-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*
Example 1 — Phosphorylation Identification by MS/MS
500 600 700 800 900 1000 1100 1200 1300 1400 1500m/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
214.18(103+111)C-DMPO
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)
Example 3 — Identification of DMPO Adduct by MS/MS
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: 309AALEFEDGMYANLGIGIPLLASNFISPNMTVHLQSENGVLGLGPYPLK356Chymotrypsin: 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.AAA176-187 505.9445 1514.8118 1514.8 4.36 3 59 K.QVKKEDIEKENR.EAA188-205 678.3385 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
Abundan
ce
27.80
27.9327.74
27.69 27.96
28.01
27.61 28.06
27.55
27.5028.13
27.4428.2127.3828.3627.3028.56
27.19 29.0725.9520.90 30.13 31.4825.54 33.67 36.0434.17 38.6538.1521.45 23.8222.8927.80
27.8227.74
27.66
27.96
27.6128.01
27.55
28.0627.50 39.6035.9128.1336.0927.44 39.55
27.3628.21 31.90 32.0927.25 39.4936.2531.7628.6427.06 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
Relat
ive A
bund
ance
733.45
620.39507.76
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
Relativ
e Abund
ance
35.88
42.9541.90
49.2343.6649.07
44.6735.9940.05
40.1536.06
40.20 45.5136.2728.36
28.2128.13
28.54 34.20 36.46 38.26 46.3933.80 46.5832.0428.6728.08 32.09 36.57 47.1631.93
24.09 27.55 47.4728.9023.8521.16 29.16
41.95
42.09 44.90
35.9545.16
44.8141.8635.82 40.13 43.30 49.3828.69 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
Abun
danc
e
733.44
620.39
832.51
507.74
416.77
460.36
300.06413.10
201.05
359.31173.20 573.18
674.27255.09
407.74555.09 656.23 715.43602.35213.08 498.76 814.46545.24 858.31310.81262.26 430.13 771.31689.49 886.44
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
Rel
ativ
e A
bund
ance
581.28
359.31
470.22
460.28
757.45545.38 658.34
413.05
402.85328.66299.98 631.30201.02 446.40
594.32173.19388.76 517.26255.10 671.51 730.39229.03 781.32702.58
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
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
LTQ DATA—Fresh Sample
Start Observed Mr(expt) Mr(calc) Delta Score Peptide
92-112 976.3 1950.5854 1949.856 0.729 108 K.SVSTPSEAGSQDSGDGAVGSR.T92-112 976.44 1950.8654 1950.84 0.025 94 K.SVSTPSEAGSQDSGDGAVGSR.T + Deamidated (NQ)
92-112 651.53 1951.5682 1950.84 0.728 66 K.SVSTPSEAGSQDSGDGAVGSR.T + Deamidated (NQ)
92-112 651.62 1951.8382 1950.84 0.998 81 K.SVSTPSEAGSQDSGDGAVGSR.T + Deamidated (NQ)92-112 1016.28 2030.5454 2030.807 -0.261 86 K.SVSTPSEAGSQDSGDGAVGSR.T + Deamidated (NQ); Phospho (ST)
92-112 678 2030.9782 2030.807 0.172 59 K.SVSTPSEAGSQDSGDGAVGSR.T + Deamidated (NQ); Phospho (ST)
Sequence Coverage 39%; T95 was phosphorylatd
1 MSDKSELKAE LERKKQRLAQ IREEKKRKEE ERKKKETDQK KEAVAPVQEE51 SDLEKKRREA EALLQSMGLT PESPIVFSEY WVPPPMSPSS KSVSTPSEAG
101 SQDSGDGAVG SRTLHWDTDP SVLQLHSDSD LGRGPIKLGM AKITQVDFPP151 REIVTYTKET QTPVMAQPKE DEEEDDDVVA PKPPIEPEEE KTLKKDEEND201 SKAPPHELTE EEKQQILHSE EFLSFFDHST RIVERALSEQ INIFFDYSGR251 DLEDKEGEIQ AGAKLSLNRQ FFDERWSKHR VVSCLDWSSQ YPELLVASYN301 NNEDAPHEPD GVALVWNMKY KKTTPEYVFH CQSAVMSATF AKFHPNLVVG351 GTYSGQIVLW DNRSNKRTPV QRTPLSAAAH THPVYCVNVV GTQNAHNLIS401 ISTDGKICSW SLDMLSHPQD SMELVHKQSK AVAVTSMSFP VGDVNNFVVG451 SEEGSVYTAC RHGSKAGISE MFEGHQGPIT GIHCHAAVGA VDFSHLFVTS501 SFDWTVKLWT TKNNKPLYSF EDNADYVYDV MWSPTHPALF ACVDGMGRLD551 LWNLNNDTEV PTASISVEGN PALNRVRWTH SGREIAVGDS EGQIVIYDVG
601 EQIAVPRNDE WARFGRTLAE INANRADAEE EAATRIPA
Detected 30 times!!!
Orbitrap XL Data---Two Days old, Stored at 4C
Start Observed Mr(expt) Mr(calc) ppm Score Peptide92-112 975.9321 1949.8496 1949.8563 -3.43 138 K.SVSTPSEAGSQDSGDGAVGSR.T92-112 975.9351 1949.8556 1949.8563 -0.36 149 K.SVSTPSEAGSQDSGDGAVGSR.T92-112 975.9354 1949.8562 1949.8563 -0.052 149 K.SVSTPSEAGSQDSGDGAVGSR.T92-112 975.9377 1949.8608 1949.8563 2.33 143 K.SVSTPSEAGSQDSGDGAVGSR.T92-112 975.9377 1949.8609 1949.8563 2.39 130 K.SVSTPSEAGSQDSGDGAVGSR.T92-112 975.9386 1949.8626 1949.8563 3.27 125 K.SVSTPSEAGSQDSGDGAVGSR.T92-112 975.9403 1949.8661 1949.8563 5.02 142 K.SVSTPSEAGSQDSGDGAVGSR.T92-112 975.9407 1949.8668 1949.8563 5.39 135 K.SVSTPSEAGSQDSGDGAVGSR.T
Sequence Coverage 53%; No Phosphorylation Detected
1 MSDKSELKAE LERKKQRLAQ IREEKKRKEE ERKKKETDQK KEAVAPVQEE51 SDLEKKRREA EALLQSMGLT PESPIVFSEY WVPPPMSPSS KSVSTPSEAG
101 SQDSGDGAVG SRTLHWDTDP SVLQLHSDSD LGRGPIKLGM AKITQVDFPP151 REIVTYTKET QTPVMAQPKE DEEEDDDVVA PKPPIEPEEE KTLKKDEEND201 SKAPPHELTE EEKQQILHSE EFLSFFDHST RIVERALSEQ INIFFDYSGR251 DLEDKEGEIQ AGAKLSLNRQ FFDERWSKHR VVSCLDWSSQ YPELLVASYN301 NNEDAPHEPD GVALVWNMKY KKTTPEYVFH CQSAVMSATF AKFHPNLVVG351 GTYSGQIVLW DNRSNKRTPV QRTPLSAAAH THPVYCVNVV GTQNAHNLIS401 ISTDGKICSW SLDMLSHPQD SMELVHKQSK AVAVTSMSFP VGDVNNFVVG451 SEEGSVYTAC RHGSKAGISE MFEGHQGPIT GIHCHAAVGA VDFSHLFVTS501 SFDWTVKLWT TKNNKPLYSF EDNADYVYDV MWSPTHPALF ACVDGMGRLD551 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*
YKVPQLEIVPNsAEERAlpha 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 CO
NH
R1
HC C
ONH
R2
HC C
ONH
Rn-1
HC 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
FS(PO4)ADYHSHLDSLSKPSEYSDLK
200 300 400 500 600 700 800 900 1000 1100 1200 1300m/z
CID
ETD
244.15z2
332.07 (252.13+80)
c2
359.20z3
403.18c3
446.25z4
518.17c4
609.31z5
681.24c5
738.34z6 818.37
c6
825.38z7
905.32c7
1042.45c8
1137.532+
z20
1222.032+
z21
1103.022+
z19963.972+
z171239.742+
c211182.492+
c20
1081.732+
c18
999.862+
c17
652.664+
MH4+
869.404+
MH3+
837.113+
MH-H3PO43+
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 sequenceMASCOT (trypsin OR no enzyme)
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
254V L S G E Q N M M T K264
200 300 400 500 600 700 800 900 1000 1100 1200m/z
1000
2000
3000
4000
5000
Inte
nsity
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 1600m/z
250
500
750
1000
1250
1350
Inte
nsity
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
“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???
• Only Protein Modification Requiring Detailed Structural Characterization (sugarchain/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 Improve the MSMS Identification