-Equipment: - 4000QTRAP AB Sciex - Tempo-nano MDLC -Protein quantitation: µg/µL, n=3 Cell lineLowry (DC)Reported MCF-75.8 ± 0.55 CCD187.2 ± 0.17 Ramos2.3 ± 0.23.95 -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 0982 758020 906040 921090 1021090 104982 115982
-MRM evaluation, specifity and verification a) manual revision of transitions -coelution of different transitions corresponding to the same peptide -good signal intensity and S/N Fructose-b aldolase GILAADESTGSIAK 666.9/907.4 +2/y9 666.9/978.5 +2/y10 666.9/1045.5 +2/y11 RT: 39,9 min
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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