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