Meeting S-HPP Chr 16 La Cristalera, 28-29 August 2012 MRM Group Zaragoza IACS Irene Orera and Sílvia Barceló-Batllori
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Meeting S-HPP Chr 16 La Cristalera, 28-29 August 2012MRM Group Zaragoza IACS
Irene Orera and Sílvia Barceló-Batllori
- Equipment - Protein quantitation- Digestion
- MRM Strategy: - MRM transition selection- MRM transition detection in samples- MRM evaluation, specifity and verification (MSMS)- MRM optimization (CE)- LOD, LOQ and reproducibility
- Limitations
- Equipment:- 4000QTRAP AB Sciex- Tempo-nano MDLC
- Protein quantitation: µg/µL, n=3
Cell line Lowry (DC) Reported
MCF-7 5.8 ± 0.5 5
CCD18 7.2 ± 0.1 7
Ramos 2.3 ± 0.2 3.95
- Protein digestion: In solution digestion:
-200mM DTT 30 min 37ºC-200mM IAA 30 min ambient temperature in dark-Trypsin gold (promega) 0.1 µg/µL in 25 mM amonium bicarbonate: protein ratio 1:20, digestion o.n. 37ºC.- Stop with formic acid and adjust pH<6
MRM Strategy: - MRM transition selection:
- MRM pilot 2.0 : -initial peptide selection: 12 – 35 aa, no Met no Cys, 0 missed cleavage, 10 peptides per protein, +2 and +3 charge-automatic fragment selection of 5 highest intensity of y ions (10 ideally)
- SRM Atlas
- MRM transition detection in samples:- HPLC:
- Trap column Acclaim PepMap 100 nano 100 um ID x 2 cm- Column Acclaim PepMap 100 nano 75 um x 15 cm- 3ug of protein injected- 90 min gradient (mobile phase A: 2% ACN, 0.1% Formic Acid, mobile
phase B: 98% ACN, 0.1% Formic Acid
MRM Strategy: -HPLC: 90 min gradient (mobile phase A: 2% ACN, 0.1% Formic Acid, mobile phase B: 98% ACN, 0.1% Formic Acid
-MS:- Ion source: nano ESI, 2800V, CUR 20psi, GS1 20psi, IHT 150ºC, DP
80V- 200 transitions/method, dwell time 50 msec- MIDAS: MRM + IDA + 2EPIs
Time (min) %A %B
0 98 2
75 80 20
90 60 40
92 10 90
102 10 90
104 98 2
115 98 2
- MRM evaluation, specifity and verificationa) manual revision of transitions
-coelution of different transitions corresponding to the same peptide-good signal intensity and S/N
Fructose-b aldolaseGILAADESTGSIAK666.9/907.4 +2/y9666.9/978.5 +2/y10666.9/1045.5 +2/y11RT: 39,9 min
Fructose-b aldolaseGILAADESTGSIAK666.9/907.4 +2/y9666.9/978.5 +2/y10666.9/1045.5 +2/y11RT: 39,9 min
b) proteotypic peptides: Blast http://web.expasy.org/blast/ and NCBI blast without gaps 100% identity
transicionProteina Peptido ion padre ion hijo
HBA1hemoglobin, alpha 1 [Source:HGNC Symbol;Acc:4823] -
ALDOAaldolase A, fructose-bisphosphate [Source:HGNC Symbol;Acc:414] GILAADESTGSIAK 666,85 978,47
666,85 1049,51 666,85 907,44 C[CAM]PLLKPWALTFSYGR 904,98 1197,61 904,98 1325,7 GVVPLAGTNGETTTQGLDGLSER 1136,57 1277,63 1136,57 1463,7 1136,57 1406,68 YTPSGQAGAAASESLFVSNHAY 1114,52 1324,62 1114,52 1253,58
ITGAM
integrin, alpha M (complement component 3 receptor 3 subunit) [Source:HGNC Symbol;Acc:6149] FGDPLGYEDVIPEADR 896,92 1043,5
896,92 1263,59
DHX38
DEAH (Asp-Glu-Ala-His) box polypeptide 38 [Source:HGNC Symbol;Acc:17211] -
GLG1golgi glycoprotein 1 [Source:HGNC Symbol;Acc:4316] ADIFVDPVLHTAC[CAM]ALDIK 999,52 1240,67
999,52 1337,72
AMFRautocrine motility factor receptor [Source:HGNC Symbol;Acc:463] SVGEAHSVSPPPR 660,34 947,51
660,34 876,47
TBL3transducin (beta)-like 3 [Source:HGNC Symbol;Acc:11587] -
NOMO2NODAL modulator 2 [Source:HGNC Symbol;Acc:22652] GAYSVGPLHSDLEYTVTSQK 1076,53 1407,68
1076,53 1270,62 VLNGPEGDGVPEAVVTLNNQIK 1132,1 1481,83 1132,1 1424,81
NUP93nucleoporin 93kDa [Source:HGNC Symbol;Acc:28958] -
TAF1C
TATA box binding protein (TBP)-associated factor, RNA polymerase I, C, 110kDa [Source:HGNC Symbol;Acc:11534] SQQHTPVLSSSQPLR 832,94 986,56
832,94 1083,62
STX1Bsyntaxin 1B [Source:HGNC Symbol;Acc:18539] -
CDH13
cadherin 13, H-cadherin (heart) [Source:HGNC Symbol;Acc:1753] EFQATVEEGAVGVIVNLTVEDK 1174,1 1413,79
1174,1 1356,77
ABAT
4-aminobutyrate aminotransferase [Source:HGNC Symbol;Acc:23] EDLLNNAAHAGK 626,82 895,47
626,82 1008,56 ALLTGLLDLQAR 642,39 885,52 642,39 986,56
DDX19B
DEAD (Asp-Glu-Ala-As) box polypeptide 19B [Source:HGNC Symbol;Acc:2742] ISEQIVIGTPGTVLDWC[CAM]SK 1052,04 1162,56
1052,04 1320,63
CDH15
cadherin 15, type 1, M-cadherin (myotubule) [Source:HGNC Symbol;Acc:1754] -
ITGAL
integrin, alpha L (antigen CD11A (p180), lymphocyte function-associated antigen 1; alpha polypeptide) [Source:HGNC Symbol;Acc:6148] -
SETD1A
SET domain containing 1A [Source:HGNC Symbol;Acc:29010] QDTPSSFGQFTPQSSQGTPYTSR 835,38 996,47
835,38 1211,57
RPS15A
ribosomal protein S15a [Source:HGNC Symbol;Acc:10389] -
CBFB
core-binding factor, beta subunit [Source:HGNC Symbol;Acc:1539]
SEIAFVATGTNLSLQFFPASWQGEQR 962,15 1205,57
962,15 1352,64VPS35
vacuolar protein sorting 35 homolog (S. cerevisiae) [Source:HGNC Symbol;Acc:13487] AQLAAITLIIGTFER 808,97 1049,6
808,97 1162,68 FTLPPLVFAAYQLAFR 927,52 1086,57 927,52 1185,64 927,52 1298,73 LFDIFSQQVATVIQSR 926,5 1129,63 926,5 1216,66 926,5 1363,73 VADLYELVQYAGNIIPR 967,52 1243,72 967,52 1372,76
Transitions detected in Ramos digest
In progress:
- MRM optimization (CE)
- peptide/transition verification/validation/identification (MSMS)
- LOD, LOQ and reproducibility
Limitations-Validation and specificity of MRM transitions by MSMS/MIDAS
(MRM Initiated Detection and Sequencing) difficult due to sample complexity and low sensitivity of our MS equipment.
-In highly complex samples like the ones we are dealing, it is highly unlikely that the peptide corresponding to the transition will be within the most intense ones and it will be fragmented ……
Alternatives: - validate MSMS in shot-gun high sensitive equipment (orbis or
similars)- SILAC labeling of cell culture, for transition/retention time specifity - aqua-synthetic peptides
Ebhardt, Sabidó et al, Proteomics2012
- cell culture SILAC label- peptide identification with light
synthetic peptides added to heavy endogenous peptides
- for transition/retention time specifity and transition order and relative intensity, allows to eliminate interfering signals
- heavy synthetic peptides (aqua) for quantitation
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