PEPTIDE SYNTHESIS in targeted proteomics

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


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


Peptide selection



PTP prediction


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


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.



PTP prediction


Peptide selection


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.



PTP prediction


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


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


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


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


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


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


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


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


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


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


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


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

PEPTIDE SYNTHESIS in targeted proteomics

Documents

Transcript of PEPTIDE SYNTHESIS in targeted proteomics