Spanish Human Proteome Project (sHPP). Proyecto SRM CIMA. María I. Mora, Fernando J. Corrales...

Spanish Human Proteome Project (sHPP).

Proyecto SRM CIMA.

María I. Mora, Fernando J. CorralesLaboratorio de Proteómica, CIMA, Universidad de Navarra

Proteína de interésSecuencia

Datos MS/MS

Crear Método MRM

Péptidos proteotípicos

Peptide verification

Crear Método MRM “optimizado”

Final Method Crear Método MRM “definitivo” (Scheduled)

Transiciones

Estrategia general

*Adquirir

*Evaluar

*Optimizar!!!

*Adquirir

*Evaluar

ensembl_gene_id external_gene_idACC Uniprot descriptionENSG00000167978 SRRM2 Q9UQ35 serine/arginine repetitive matrix 2 [Source:HGNC Symbol;Acc:16639]ENSG00000140750 ARHGAP17 Q68EM7 Rho GTPase activating protein 17 [Source:HGNC Symbol;Acc:18239]ENSG00000177238 TRIM72 Q6ZMU5 tripartite motif containing 72 [Source:HGNC Symbol;Acc:32671]ENSG00000090861 AARS P49588 alanyl-tRNA synthetase [Source:HGNC Symbol;Acc:20]ENSG00000135723 FHOD1 Q9Y613 formin homology 2 domain containing 1 [Source:HGNC Symbol;Acc:17905]ENSG00000166501 PRKCB P05771 protein kinase C, beta [Source:HGNC Symbol;Acc:9395]ENSG00000079616 KIF22 Q14807 kinesin family member 22 [Source:HGNC Symbol;Acc:6391]ENSG00000171490 RSL1D1 O76021 ribosomal L1 domain containing 1 [Source:HGNC Symbol;Acc:24534]ENSG00000177200 CHD9 Q3L8U1 chromodomain helicase DNA binding protein 9 [Source:HGNC Symbol;Acc:25701]ENSG00000166848 TERF2IP Q9NYB0 telomeric repeat binding factor 2, interacting protein [Source:HGNC Symbol;Acc:19246]ENSG00000070770 CSNK2A2 P19784 casein kinase 2, alpha prime polypeptide [Source:HGNC Symbol;Acc:2459]ENSG00000065457 ADAT1 Q9BUB4 adenosine deaminase, tRNA-specific 1 [Source:HGNC Symbol;Acc:228]ENSG00000015413 DPEP1 P16444 dipeptidase 1 (renal) [Source:HGNC Symbol;Acc:3002]ENSG00000166747 AP1G1 O43747 adaptor-related protein complex 1, gamma 1 subunit [Source:HGNC Symbol;Acc:555]ENSG00000141012 GALNS P34059 galactosamine (N-acetyl)-6-sulfate sulfatase [Source:HGNC Symbol;Acc:4122]ENSG00000100726 TELO2 Q9Y4R8 TEL2, telomere maintenance 2, homolog (S. cerevisiae) [Source:HGNC Symbol;Acc:29099]ENSG00000103381 CPPED1 Q9BRF8 calcineurin-like phosphoesterase domain containing 1 [Source:HGNC Symbol;Acc:25632]ENSG00000157106 SMG1 Q96Q15 smg-1 homolog, phosphatidylinositol 3-kinase-related kinase (C. elegans) [Source:HGNC Symbol;Acc:30045]ENSG00000122299 ZC3H7A Q8IWR0 zinc finger CCCH-type containing 7A [Source:HGNC Symbol;Acc:30959]ENSG00000069345 DNAJA2 O60884 DnaJ (Hsp40) homolog, subfamily A, member 2 [Source:HGNC Symbol;Acc:14884]

20 proteínas asignadas a CIMA

Prueba preliminar en células Huh7

Preparación de la muestra

a) Lisis: en Urea 7M / Thiourea 2M / CHAPS 4% / DTT 40mM

b) Precipitación: cleanup kit (BioRad) Pellet resuspendido en Bicarbonato de Amonio 50mM

PROBLEMA: Resuspensión!!!!

c) Digestión: Reducción 10mM DTT, 56ºC, 30’

Alquilación 28mM IAA, RT, 20’

Digestión 1:50, 37ºC, o/n

d) A QTRAP 5500: 1 ug / run

Transiciones: Definidas por MRM pilot

3

2

4

Ignore if:

-KK/ KR/ RR/ RK (end)

-end of sequence

and

1

Métodos MRM individuales

Péptidos Transiciones pept - transic dwell time (msec) scan time (s)

A 20 - 101 15 2.01B 28 - 128 11 2.04C 23 -130 10 1.95

ARHGAP17 Q68EM7 10 55 1 A 10 - 55 30 1.92TRIM72 Q6ZMU5 11 61 1 A 11 - 61 28 2.01AARS P49588 20 109 1 A 20 - 109 13 1.96

A 13 -71 23 1.98B 17 - 90 18 2.06

PRKCB P05771 13 73 1 A 13 - 73 20 1.82KIF22 Q14807 18 98 1 A 18 - 98 15 1.96

RSL1D1 O76021 10 52 1 A 10 -52 32 1.91A 15 -80 20 1.99B 21 -112 13 2.01C 19 -98 15 1.96

TERF2IP Q9NYB0 12 68 1 A 12 -68 25 1.97CSNK2A2 P19784 9 48 1 A 9 - 48 35 1.91

ADAT1 Q9BUB4 13 71 1 A 13 - 71 23 1.98DPEP1 P16444 10 59 1 A 10 - 59 29 2.01AP1G1 O43747 10 55 1 A 10 - 55 30 1.92GALNS P34059 11 60 1 A 11 - 60 28 1.97

A 14 - 75 20 1.87B 12 -65 25 1.94

CPPED1 Q9BRF8 8 49 1 A 8 - 49 35 1.95A 20 - 105 14 1.99B 23 - 116 13 2.08C 21 - 110 14 2.08D 19 - 97 16 2.03

ZC3H7A Q8IWR0 20 110 1 A 20 - 110 13 1.97DNAJA2 O60884 7 39 1 A 7 -39 45 1.94

PROTEINA Nº métodos

SRRM2 Q9UQ35 71 359 3

SMG1 Q96Q15

FHOD1 Q9Y613

CHD9 Q3L8U1

TELO2 Q9Y4R8

30 97

55 290

26 140

83 428

3

2

2

4

Condiciones LC

Pickup: 2ul

Loading: 8min (3ul/min)

Gradiente: 60 min (300nl/min)

Ejemplos

MRMpilotAnalyst

Proteina:

AARS

Peptido:

ALNEALK / 379.72

TR:

22.4’

+MRM (109 pairs): Exp 1, 22.400 min from Sample 1 (PepVerif_AARS_A_60min_1ul_2012 15 06) of PepVerif_AARS_A_60min_1ul_20... Max. 5.0e4 cps.

685.040/1070.600685.040/1141.640

514.030/686.360379.720/574.320

379.720/687.400379.720/612.340

379.720/499.250379.720/460.280

379.720/428.210438.720/761.420

438.720/575.310438.720/662.350

438.720/730.340

Q1/Q3 Masses, Da

0.0

2000.0

4000.0

6000.0

8000.0

1.0e4

1.2e4

1.4e4

1.6e4

1.8e4

2.0e4

2.2e4

2.4e4

2.6e4

2.8e4

3.0e4

3.2e4

3.4e4

3.6e4

3.8e4

4.0e4

4.2e4

4.4e4

4.6e4

4.8e4

5.0e4

Inte

ns

ity, c

ps

y5

y6 b6

b5

y4

b4

Programadas: 6

Observadas: 6

Programadas: 6

Observadas: 5

AnalystMRMpilot

Proteina:

TRIM72

Peptido:

ILEAHVEAK / 505.29

TR:

20.6’

+MRM (61 pairs): Exp 1, 20.605 min from Sample 1 (PepVerif_TRIM72_A_60min_1ul_2012 15 06) of PepVerif_TRIM72_A_60min_1ul... Max. 8036.0 cps.

553.990/927.580 505.290/654.360 505.290/783.400 337.190/654.360 505.290/896.480 505.290/863.460 505.290/583.320Q1/Q3 Masses, Da

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

5500

6000

6500

7000

7500

8000

Inte

ns

ity, c

ps

*

y6

y7

y8

b8

y5

Analyst

MRMpilot

Proteina:

TELO2

Peptido:

FDRPLVTFDLLGEDQLVLGR / 768.42

TR:

40.34’

Programadas: 5

Observadas: 5

+MRM (65 pairs): Exp 1, 40.311 min from Sample 1 (PepVerif_TELO2_B_60min_1ul_2012 15 06) of PepVerif_TELO2_B_60min_1ul_2... Max. 2800.0 cps.

593.980/1067.600890.470/1154.540

768.420/1212.690768.420/800.460

768.420/1099.610768.420/929.510

768.420/986.530609.360/791.460

609.360/920.500406.570/791.460

609.360/1033.580609.360/1146.670

Q1/Q3 Masses, Da

0

200

400

600

800

1000

1200

1400

1600

1800

2000

2200

2400

2600

2800

Inte

ns

ity, c

ps

y10

y7

y9

y8

y11

Proteina:

SRRM2

Peptido:

STTPAPK / 351.19

TR:

16.24’

Programadas: 5

Observadas: 5

Analyst

+MRM (101 pairs): Exp 1, 16.226 min from Sample 1 (PepVerif_SRRM2_A_60min_1ul_2012 15 06) of PepVerif_SRRM2_A_60min_1ul_... Max. 9.5e4 cps.

343.680/426.200789.940/959.570

789.940/1056.620526.960/959.570

526.960/734.420789.940/1185.660

351.190/412.260351.190/513.300

351.190/614.350351.190/555.280

351.190/458.220679.790/897.390

679.790/810.360679.790/1012.420

679.790/1083.450679.790/1170.490

Q1/Q3 Masses, Da

0.0

5000.0

1.0e4

1.5e4

2.0e4

2.5e4

3.0e4

3.5e4

4.0e4

4.5e4

5.0e4

5.5e4

6.0e4

6.5e4

7.0e4

7.5e4

8.0e4

8.5e4

9.0e4

9.5e4

Inte

ns

ity, c

ps

y4

y5

y6

b6

b5

AnalystMRMpilot

Proteina:

CHD9

Peptido:

TEEQVQK / 431.22

TR:

21.65’

Programadas: 5

Observadas: 4

+MRM (80 pairs): Exp 1, 21.650 min from Sample 1 (PepVerif_CHD9_A_60min_1ul_2012 15 06) of PepVerif_CHD9_A_60min_1ul_201... Max. 5.8e4 cps.

980.520/1229.700654.010/1130.630

654.010/1229.700980.520/1191.600

431.220/631.340431.220/502.300

431.220/760.380431.220/715.330

431.220/587.270421.720/581.330

421.720/728.400421.720/696.340

421.720/609.300421.720/496.220

476.230/589.260317.820/589.260

476.230/717.350

Q1/Q3 Masses, Da

0.0

5000.0

1.0e4

1.5e4

2.0e4

2.5e4

3.0e4

3.5e4

4.0e4

4.5e4

5.0e4

5.5e4

5.8e4

Inte

ns

ity, c

ps

b6

b5

y4y5

! !

Proteina:

ARGHAP17

Peptido:

DPVSAAVPAPGR / 568.81

TR:

19.85’ – 21.41’

MRMpilotAnalyst

+MRM (55 pairs): Exp 1, 19.849 min from Sample 1 (PepVerif_ARHGAP17_A_60min_1ul_2012 15 06) of PepVerif_ARHGAP17_A_60... Max. 8367.0 cps.

488.260/616.290568.810/1021.580

568.810/667.390568.810/738.430

379.540/497.280568.810/825.460

568.810/924.530446.230/463.230

446.230/578.250446.230/679.300

446.230/778.370

Q1/Q3 Masses, Da

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

5500

6000

6500

7000

7500

8000

8367

Inte

ns

ity, c

ps

y11

y7

y8

y5

y9

y10

+MRM (55 pairs): Exp 1, 21.413 min from Sample 1 (PepVerif_ARHGAP17_A_60min_1ul_2012 15 06) of PepVerif_ARHGAP17_A_60m... Max. 2.3e4 cps.

488.260/663.330488.260/791.390

488.260/862.430488.260/829.410

488.260/715.360488.260/616.290

568.810/1021.580568.810/667.390

568.810/738.430379.540/497.280

568.810/825.460568.810/924.530

446.230/463.230446.230/578.250

Q1/Q3 Masses, Da

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

2.3e4

Inte

ns

ity, c

ps

y11

y7

y8

y5y9

y10Programadas: 6

Observadas: 6

Analyst

Proteina:

DNAJA2

Peptido:

ENEVFQR /

472.73

TR:

16.3’ – 25.5’

+MRM (39 pairs): Exp 1, 16.312 min from Sample 1 (PepVerif_DNAJA2_A_60min_1ul_2012 15 06) of PepVerif_DNAJA2_A_60min_1ul... Max. 2.2e4 cps.

880.800/1127.670660.850/1224.720 472.730/815.420 472.730/549.310 472.730/678.360 315.490/549.310 472.730/770.350 472.730/642.290 931.940/1226.580931.940/1000.480Q1/Q3 Masses, Da

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e4

1.9e4

2.0e4

2.1e4

2.2e4

Inte

ns

ity, c

ps

y6

y5

y4

b6y4*

b5

+MRM (39 pairs): Exp 1, 25.533 min from Sample 1 (PepVerif_DNAJA2_A_60min_1ul_2012 15 06) of PepVerif_DNAJA2_A_60min_1u... Max. 3756.0 cps.

880.800/1070.650880.800/1127.670

660.850/1224.720472.730/815.420

472.730/549.310472.730/678.360

315.490/549.310472.730/770.350

472.730/642.290931.940/1226.580

931.940/1000.480931.940/1129.530

Q1/Q3 Masses, Da

0

200

400

600

800

1000

1200

1400

1600

1800

2000

2200

2400

2600

2800

3000

3200

3400

3600

3756

Inte

ns

ity, c

ps

y6

y4

y5

b6

b5

*

Programadas: 6

Observadas: 6 / 5

E en N-terminal

Resultados MRM (peptide verification- I)

Péptidos Transiciones Posibles péptidos num. transiciones num. transiciones programados programadas a optimizar (Huh7) observadas programadas

379.72 22.4 6 6365.2 20.7 / 22.69 2 / 5 5

738.88 37.35 / 36.18 4 / 6 7530.76 / 354.18 32.91 4 6

450.78 21.02 5 5439.24 27.15 5 6530.25 19.4 5 6407.72 20.13 / 21.2 5 / 5 5423.25 22.7 6 6430.72 27.5 5 6432.23 19.95 5 5471.24 28.24 5 5

568.81 / 379.54 19.85 / 21.41 6 / 6 6616.33 / 411.22 20.99 4 5

551.8 / 368.2 28.6 5 5493.77 20.46 / 20.77 6 / 6 6422.76 17.77 6 6429.74 18.1 5 5424.7 23.08 / 26.73 3 / 3 5

431.22 21.65 5 5462.72 / 308.82 16.7 6 7

372.7 16.94 / 18.86 / 20.37 5 / 2 / 4 5539.3 24.4 4 5

503.26 / 335.85 27.75 5 6386.71 19.47 5 5722.77 28.39 4 5

478.78 / 319.52 21.4 / 26.8 / 27.3 5 / 4 / 2 6

Q1 (z2 / z3) TR observadoProteína

55 290 11

10 55 3

10 55 4

20 109 5

13 71 4

CHD9

AARS

ADAT1

AP1G1

ARHGAP17

Resultados MRM (peptide verification- II)

Péptidos Transiciones Posibles péptidos num. transiciones num. transiciones programados programadas a optimizar (Huh7) observadas programadas

CPPED1 8 49 1 551.28 / 367.86 28.05 7 7932.93 / 622.29 32.25 3 5

351.71 16.28 5 5725.4 / 483.94 19.86 5 6

762.91 / 508.94 35.3 5 6886.45 / 591.3 29.38 4 5

472.73 / 315.49 16.3 / 25.5 6 / 5 6569.29 / 379.86 19.1 7 7

411.71 22.9 5 5464.25 / 309.83 17.3 6 6488.74 / 326.16 16.47 6 6536.78 / 358.19 25.51 5 6661.34 / 441.23 26.02 5 5

454.23 15.8 5 5453.76 / 302.84 23.15 5 6506.27 / 337.85 16.5 6 6

436.25 21.45 5 5351.22 19.5 4 5438.72 15.4 / 16.86 6 / 6 6422.71 18.16 / 20.68 6 / 6 6

782.76 / 587.32 32.16 4 5435.23 20.2 5 6

798.45 / 532.64 28.62 4 5414.22 22.1 4 6434.78 16.85 5 5

PRKCB 13 73 0

Proteína Q1 (z2 / z3) TR observado

10 52 2

11 60 3

18 98 3

10 59 3

30 97 5

9 48 5

7 39 3

FHOD1

GALNS

KIF22

RSL1D1

CSNK2A2

DNAJA2

DPEP1

Resultados MRM (peptide verification- III)

Péptidos Transiciones Posibles péptidos num. transiciones num. transiciones programados programadas a optimizar (Huh7) observadas programadas

396.24 20.18 / 22.14 / 22.18 3 / 3 / 3 5453.75 24.97 5 5579.29 19.05 5 5404.24 19.26 5 5376.2 18.95 5 5

370.24 16.55 5 5552.78 / 368.86 25.48 2 5

429.74 20.66 6 6598.8 17.22 5 5

615.81 / 410.87 27.41 5 6493.29 / 329.19 27.11 5 5

449.25 29.05 4 5920.95 / 614.3 34.1 5 5

393.7 15.81 4 4351.19 16.24 5 5

464.25 / 309.84 21.54 2 5759.02 / 569.52 35.18 2 4

389.2 17.15 2 4460.72 / 307.48 16.26 5 6

351.18 15.66 / 19.26 5 / 4 5414.75 17.52 5 5

650.86 / 434.25 28.44 4 6486.27 16.3 4 6467.75 19.8 5 5

729.4 / 486.6 24.2 4 5438.77 25.5 3 5540.37 27.52 4 6768.42 40.34 5 5510.77 27.5 3 5

TERF2IP 12 68 0TRIM72 11 61 1 505.29 / 337.19 20.59 5 6

369.19 15.4 / 18.4 5 / 6 6433.71 15.6 5 5

594.81 / 396.88 28.05 5 6417.76 17.98 5 5

465.77 / 310.85 23.17 6 6670.82 / 447.55 16.14 4 6

Q1 (z2 / z3) TR observado

20 110 6

Proteína

71 359 12

26 140 5

83 428 12SMG1

SRRM2

TELO2

ZC3H7A

Proteína de interésSecuencia

Datos MS/MS

Crear Método MRM

Péptidos proteotípicos

Peptide verification

Crear Método MRM “optimizado”

Final Method Crear Método MRM “definitivo” (Scheduled)

Transiciones

Estrategia general

*Adquirir

*Evaluar

*Optimizar!!!

*Adquirir

*Evaluar

Condiciones QTRAP 5500

MRM- Peptide Verification