peptide synthesis in targeted proteomics proteomics facility – national centre of biotechnology...
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PEPTIDE SYNTHESISin targeted proteomics
Proteomics Facility – National Centre of Biotechnology
SpHPP
Manuel Lombardía Uría
Peptide synthesis for targeted proteomics
Selection and design of peptides for each protein of interest:
- essential process for the success of SRM/MRM experiments
- selection based on experimental and theoretical criteria
- obtention of optimal proteotypic peptides
Chemical synthesis
Experimental validation of proteotypic peptides
Heavy isotope peptide synthesis
Stages:
Peptide selection
Proteins of interest
Known Unknown
ProteinAccession
numberName Protein
Accession number
Name
P02795 MT2_HUMAN Metallothionein-2 Q96M29 TEKT5_HUMAN Tektin-5Q58EX7 PKHG4_HUMAN Puratrophin-1 Q96MC5 CP045_HUMAN Uncharacterized protein C16orf45
Q01726 MSHR_HUMANMelanocyte-stimulating hormone receptor
Q14028 CNGB1_HUMANCyclic nucleotide-gated cation channel beta-1
Q14764 MVP_HUMAN Major vault protein P23975 SC6A2_HUMANSodium-dependent noradrenaline transporter
Q0VD83 APOBR_HUMAN Apolipoprotein B receptor Q8NC67 NETO2_HUMAN Neuropilin and tolloid-like protein 2
Q6EMK4 VASN_HUMAN Vasorin Q8IZ96 CKLF1_HUMANCKLF-like MARVEL transmembrane domain-containing protein 1
O14983 AT2A1_HUMANSarcoplasmic/endoplasmic reticulum calcium ATPase 1
P33527 MRP1_HUMANMultidrug resistance-associated protein 1
P22695 QCR2_HUMANCytochrome b-c1 complex subunit 2, mitochondrial
P60510 PP4C_HUMANSerine/threonine-protein phosphatase 4 catalytic subunit
P55287 CAD11_HUMAN Cadherin-11 Q49A26 GLYR1_HUMAN Putative oxidoreductase GLYR1 Q4KMP7 TB10B_HUMAN TBC1 domain family member 10B Q14019 COTL1_HUMAN Coactosin-like protein
Q9NXV2 KCTD5_HUMANBTB/POZ domain-containing protein KCTD5
Q9Y221 NIP7_HUMAN60S ribosome subunit biogenesis protein NIP7 homolog
P55259 GP2_HUMANPancreatic secretory granule membrane major glycoprotein GP2
O75150 BRE1B_HUMAN E3 ubiquitin-protein ligase BRE1B
Q2VPK5 CTU2_HUMANCytoplasmic tRNA 2-thiolation protein 2
Q8N9N5 BANP_HUMAN Protein BANP
Assigned proteins
Peptide selection
Proteins of interest
Experimental evidence
Tryptic peptide observation by LC-MS/Ms analysis in biological samples
PTP prediction
Candidate proteotypic peptides resulting from “in silico” digestion
Peptide selection
Proteins of interest
Peptide selection
Experimental evidence
Tryptic peptide observation by LC-MS/Ms analysis in biological samples
PTP prediction
Candidate proteotypic peptides resulting from “in silico” digestion
Peptide selection criteria
• Unique for each protein: Uniprot/Swiss prot searching (BLAST)
• At least 3-4 peptides for protein
• No missed cleavages
• Containing 6-20 residues
• Low hydrofobicity: values from 10 to 40 according to the SSRCalc algorithm
• Avoid aa containing potential modification sites:
- Met and Trp undergo rapid oxidation
- Asn paired with G or P can be deamidated
- Gln or Glu at N-term undergo cyclization to pyroglutamic. Also C when carbamidomethylated
- Asn at N-term: protecting group can be difficult to remove during cleavage
- Asp paired with Gly, Pro or Ser can be hydrolised under acidic conditions
- Acidic aa at position P2 or P2’ promotes missed cleavage
- Asn in glycosilation consensus sequences (Asn-X-Ser/Thr)
- His residues close to either N or C terminus can undergo charge supression
Peptide selection
Proteins of interest
Peptide selection
Búsqueda de evidencias en BD
Not all the possible peptides can be experimentally observed. Select the most observed peptides in repositories such as GPMDB or SMS atlas.
Experimental evidence
Tryptic peptide observation by LC-MS/Ms analysis in biological samples
PTP prediction
Candidate proteotypic peptides resulting from “in silico” digestion
Peptide selection
Proteins of interest
Peptide selection
Peptide synthesis
Búsqueda de evidencias en BD
Not all the possible peptides can be experimentally observed. Select the most observed peptides in repositories such as GPMDB or SMS atlas.
Experimental evidence
Tryptic peptide observation by LC-MS/Ms analysis in biological samples
PTP prediction
Candidate proteotypic peptides resulting from “in silico” digestion
Peptide synthesis
• Equipment: Authomatic synthesizer Multipep (Intavis AG)
• Method: Solid phase peptide synthesis (SPPS) using polymeric resins proloaded and FMOC
• Format: 96 minicolumn plates at a scale of 1-10 µmol
• Peptide purification by semi-preparative HPLC
• Resins preloaded with isotopically labeled Arg and Lys for the synthesis of peptides containing heavy Arg or Lys at the C-terminus
• Heavy peptide quantification by amino acid analysis or fluorescence spectroscopy (commercial kit)
Synthesized peptides crude or purified
Validation by SRM/MRM experiments
Optimal proteotypic peptides
Heavy peptides synthesis
MALDI-TOF MS testing
Peptide validation
699.0 960.8 1222.6 1484.4 1746.2 2008.0
Mass (m/z)
2.7E+4
0
10
20
30
40
50
60
70
80
90
100
% In
tens
ity
4700 Reflector Spec #1 MC[BP = 858.5, 26857]
858.
4847
880.
4660
902.
4490
829.
4712
1515
.771
0
706.
2161
730.
4156
801.
4609
1031
.568
8
758.
3929
957.
5526
986.
5452
862.
4854
929.
5261
1054
.324
1
1741
.975
2
1259
.652
6
1537
.746
6
1614
.829
1
699.0 960.8 1222.6 1484.4 1746.2 2008.0
Mass (m/z)
3.3E+4
0
10
20
30
40
50
60
70
80
90
100%
Inte
nsity
4700 Reflector Spec #1 MC[BP = 858.5, 33418]
858.
4755
880.
4547
920.
3995
986.
5338
812.
4637
730.
4085
706.
2107
959.
5228
1212
.636
2
844.
4619
872.
4819
1059
.433
1
773.
3882
1029
.507
8
1151
.416
0
1098
.571
7
Péptido 1 crudo
Péptido 1 purificado
699.0 960.8 1222.6 1484.4 1746.2 2008.0
Mass (m/z)
4.3E+4
0
10
20
30
40
50
60
70
80
90
100
% In
tens
ity
4700 Reflector Spec #1 MC[BP = 1102.6, 43366]11
02.5
967
1201
.668
6
1146
.574
6
1124
.574
3
1084
.590
3
1058
.605
8
1247
.651
0
1173
.632
0
1349
.627
6
987.
5719
1455
.668
0
1015
.564
5
1274
.628
9
1323
.666
1
872.
4329
1223
.645
3
1614
.776
9
706.
2114
1426
.883
1
728.
2209
1719
.802
5
1913
.779
5
1670
.836
9
812.
2496
916.
4207
1860
.945
8
834.
2737
1805
.833
9
1745
.913
1
699.0 960.8 1222.6 1484.4 1746.2 2008.0
Mass (m/z)
1.6E+4
0
10
20
30
40
50
60
70
80
90
100
% In
tens
ity
4700 Reflector Spec #1 MC[BP = 1102.6, 16056]
1102
.580
7
1124
.558
7
1084
.570
8
1158
.636
1
987.
5428
1106
.577
4
1217
.606
2
1288
.659
5
1189
.609
4
1324
.645
0
858.
4717
706.
2123
782.
3582
1134
.574
1
890.
4286
1058
.583
5
1239
.588
6
747.
4033
916.
4717
1009
.516
9
805.
3074
1349
.614
3
836.
3185
1031
.495
2
1082
.557
1
1812
.853
8
938.
4106
1668
.903
0
1261
.586
7
1376
.623
2
1555
.827
8
1416
.669
6
1707
.794
3
1458
.743
8
Péptido 1 crudo
Péptido 1 purificado
699.0 960.8 1222.6 1484.4 1746.2 2008.0
Mass (m/z)
1.9E+4
0
10
20
30
40
50
60
70
80
90
100
% In
tens
ity
4700 Reflector Spec #1 MC[BP = 974.5, 19491]
974.
4683
996.
4519
1012
.424
610
18.4
344
1056
.388
7
706.
1810
732.
1826
957.
4786
811.
3411
917.
4496
1078
.368
0
867.
3831
1016
.441
2
787.
1931
994.
4355
756.
1943
836.
2180
1103
.541
9
891.
2199
1126
.489
5
1249
.555
7
1516
.651
0
699.0 960.8 1222.6 1484.4 1746.2 2008.0
Mass (m/z)
9.7E+3
0
10
20
30
40
50
60
70
80
90
100%
Inte
nsity
4700 Reflector Spec #1 MC[BP = 841.1, 9657]
841.
0693
825.
0958
857.
0419
996.
4968
974.
5068
1012
.468
2
706.
2087
815.
6487
873.
6938
757.
6083
931.
7352
989.
7773
1129
.778
4
1085
.747
7
730.
2114
1217
.826
3
1261
.855
3
1041
.726
1
1173
.805
5
953.
6709
1305
.880
7
909.
6452
1349
.913
0
1393
.934
7
845.
0809
787.
2258
1437
.964
2
1481
.985
6
1014
.138
9
1063
.791
3
1526
.011
2
1570
.038
7
1614
.072
9
1658
.097
8
1702
.108
4
1746
.133
9
Péptido 3 crudo
Péptido 3 purificado
799.0 1040.8 1282.6 1524.4 1766.2 2008.0
Mass (m/z)
1.9E+4
0
10
20
30
40
50
60
70
80
90
100
% In
tens
ity
4700 Reflector Spec #1 MC[BP = 1608.8, 19123]
1608
.782
7
1630
.765
916
46.7
395
1652
.744
8
877.
0210
1690
.698
0
813.
2155
1591
.838
5
836.
2330
1613
.802
6
908.
9665
1712
.679
9
1564
.779
4
935.
2369
1054
.296
1
1206
.584
0
958.
2523
986.
5162
1502
.693
7
1025
.499
3
1737
.812
6
1360
.631
6
1098
.300
2
1127
.510
9
1077
.285
2
1780
.814
3
1529
.728
3
1174
.319
8
1279
.629
4
1759
.809
0
1153
.326
2
1410
.692
6
1238
.569
2
1801
.820
6
799.0 1040.8 1282.6 1524.4 1766.2 2008.0
Mass (m/z)
1.0E+4
0
10
20
30
40
50
60
70
80
90
100
% In
tens
ity
4700 Reflector Spec #1 MC[BP = 1571.8, 10217]
1571
.781
4
1630
.813
4
1587
.759
5
1646
.786
9
1608
.821
4
1549
.783
1
1591
.825
8
1726
.799
2
1668
.761
6
1612
.819
8
1704
.801
6
1553
.790
6
814.
3799
851.
4317
1296
.618
7
1072
.552
6
1532
.809
8
1442
.703
7
1643
.826
0
1094
.549
9
980.
4749
1760
.860
4
882.
4309
1502
.739
0
913.
4525
1127
.546
1
1355
.659
2
1022
.486
8
1044
.472
5
1403
.698
7
1473
.728
4
1803
.983
9
948.
4916
1887
.006
7
1155
.561
6
1381
.691
9
1001
.436
0
1226
.607
7
1865
.012
8
1264
.617
9
1827
.958
1
1324
.625
0
1190
.572
6
Péptido 4 crudo
Péptido 4 purificado
Synthesized peptides
AN Peptide sequence MW mg Yield (%) Quality
ACGGPTQELSALR 1302,4 6 46,1 +
DIAPSEEAINDR 1329,3 0,6 22,6 +
EAQSLVHFQLR 1327,5 1,4 52,7 +
VDGLLHQLTLQSNQR 1721,9 4,1 23,8 +
YISIFYALR 1145,3 2,5 21,8 +
YHSIVTLPR 1085,2 1,2 55,3 +
ACQHAQGIAR 1054,1 0,5 23,7 +
ALQPLEEGEDEEK 1486,6 1,4 47,1 +
ELPPGVEELLNK 1337,6 1,4 52,3 +
HYCTVANPVSR 1246,3 1,4 56,2 +
LFSVPDFVGDACK 1397,6 5,8 41,5 +
CGDYHPEGEAPR 1330,4 1,8 67,6 +
GNTQEDAADGEQR 1390,4 2 71,9 +
IVEEEAQEDLEGLR 1629,7 8,4 51,5 +
TEEAAESQTAGR 1249,3 0,9 36,0 +
ESHVTLASPEETR 1455,5 1,6 55,0 +
SLTLGIEPVSPTSLR 1570 3,3 21,0 +
VPLEPGPK 836 0,4 23,9 +
YGLNELPAEEGK 1319,6 1,1 41,7 +
GAPEGVIDR 913,1 1,2 65,7 +
VDQSILTGESVSVIK 1574,7 0,6 19,1 -
VGEATETALTTLVEK 1561,8 2 12,8 -
O14983
Q58EX7
Q01726
Q14764
Q0VD83
Q6EMK4
AN Peptide sequence MW mg Yield (%) Quality
DGAFAEFLR 1025,3 1,1 53,6 +
ENILFGCQLEEPYYR 1874,2 1,6 42,7 +
HIFENVIGPK 1153,4 0,5 21,7 +
TQFEDCTVLTIAHR 1633,8 3,6 22,0 +
DPDAANSPIR 1055,2 0,4 19,0 +
DTNRPLEPPSEFIVK 1741,9 1,2 34,4 +
LHSDIDSGDGNIK 1370,7 1,4 51,1 +
VEAANVHIDPK 1192,3 1,6 67,1 +/-
CQNILQGNFKPDFYLK 1928,2 3,5 18,2 +
DLVLGPSGVLQGIRPGK 1706,3 3 87,9 +
TSFFLGEVGNAAK 1340,7 1,3 48,5 +
VEQLKPYHAHK 1349,5 1,6 59,3 +
ALAAGADSPK 900,2 1,5 83,3 +
ELLEQNPGK 1027,2 1,4 68,1 +
VALVLLR 783 4,5 57,5 +
DLAEEGVLEEAEFYNITSLIK 2383,6 - 0,0 -
LCQADPDLDSDK 1319,5 0,9 34,1 +
LNVGGTYFLTTR 1341,7 1,3 48,4 +/-
TSQVPVK 757,8 0,4 4,4 +
LAANISGDK 888,2 1,1 61,9 +
LVSLGTCFGK 1024,2 2,3 22,5 +
VYYVSEK 887 0,7 39,5 +
YIGENLQLLVDRPDGTYCFR 2372,6 4,2 17,7 +/-
DPNCSSILQTEER 1491,6 0,1 3,4 +/-
NTLSLVNDFIIR 1404,6 0,1 3,6 +/-
SEVPAIDLAR 1070,3 0,8 37,4 +
STENSAGSQGCDK 1283,4 1,3 50,6 +
P55287
P33527
Q49A26
Q4KMP7
Q9NXV2
Q9Y221
P55259
AN Peptide sequence MW mg Yield (%) Quality
ETGEYR 753,8 0,5 33,2 +
GLLLATK 714,9 2,4 33,6 +
ISLEYSELQDK 1324,6 1,1 41,5 +
VTSAETK 734,8 0,9 61,2 +
AGDAFCR 738,9 1 67,7 +
DCLIEDSDDEAGQS 1496,6 - 0,0 -
EVAFYNR 898,0 1 55,7 +
IPCLLAPSVFK 1187,5 4 33,7 +
LDLVTNK 802,0 1,2 74,8 +
LDSIEAK 646,8 1,2 92,8 +
LQALEATCK 976,1 1 51,2 +
SCCSCCPVGCAK 1159,4 0,2 8,6 -
CAQGCICK 824,3 0,4 24,3 +/-
ATAAPAGAPPQPQDLEFTK 1909,0 1,7 44,5 +
NALANPLYCPDYR 1508,7 1,1 36,5 +
GSNTTSHLHQAVAK 1449,7 1,9 65,5 +
TIAQGNLSNTDVQAAK 1629,8 0,8 24,5 +/-
EILVEESNVQR 1314,7 1,1 41,8 +
CGNVAAILELDEHLQK 1752,9 - -
DFIIFEAAPQETR 1535,8 1,4 45,6 +
VDSPVTVCGDIHGQFYDLK 2092,0 1,7 40,6 +
DAQGLVLFDVTGQVR 1616,9 0,7 21,6 +
LAQDPFPLYPGEVLEK 1814,9 2,2 60,6 +
LFSVPDFVGDACK 1396,7 1,4 50,1 +
IEGEGSVLQAK 1129,6 1,2 53,1 +
Q2VPK5
Q8N9N5
O75150
P02795
P22695
P60510
Q14764
Conclusion
We can conclude from our results that our peptide synthesis method is suitable for the production of a high number of peptides with enough amount and quality for targeted proteomics applications.
WE OFFER:
-Regular Synthesis
-Heavy Peptide Sinthesis
- Coupling to carrier protein for immunization
- N-term- Acetylation
- C-term Amidation
- Ser/Thr/Tyr Phosphorilation
- N-term Acylation
- Cys palmitoylatión
- N-term Biotinilation
- Peptide Arrays up to 620 spots per membrane
Los precios 75 y 30€ (Arg y Lys) son sólo de la resina para cada péptido. Habría que añadir los 15 - 20€ por la síntesis. La cuantificación sólo se incluiría para los péptidos pesados precio? Tiempo?.
Los tiempos para péptidos crudos, depende de cuántos sean, pero p.e. para media placa (48 péptidos) serían dos días para la síntesis y una semana para pasar por HPLC y al menos un día más para liofilizar, pesar y etiquetar. De 10-15 días si todo va bien.
PRACTICAL ISSUES
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