A response curve consisting of peptides spiked into plasma was prepared as described previously (34 (link)). In brief, for the 3-MRM data set, a 9-point response curve was generated by spiking 11 synthetic peptides (representing prostate-specific antigen, horseradish peroxidase, leptin, myelin basic protein, myoglobin, aprotinin, and C-reactive protein) into digested plasma (1 μg/μL) so as to span a concentration range of 1–500 fmol/μL, with corresponding isotopically labeled (13C/15N) peptide standards added at a fixed concentration of 50 fmol/μL to all samples [referred to as study I in (34 (link))]. A 1-μL volume of each sample was analyzed in quadruplicate by LC-MRM-MS. Three data sets were acquired with a 4000 Q TRAP mass spectrometer (Applied Biosystems) at unit/unit resolution with a dwell time of 10 ms and an interscan delay time of 5 ms for each transition. One data set was acquired on a TSQ Quantum Ultra (Thermo Fisher Scientific) triple-quadrupole mass spectrometer with a 10-ms dwell time for each transition at unit/unit resolution. In all cases, only data from 10 peptides were analyzed, owing to insufficient detection of the 11th peptide.
For the 5-MRM data set, an equimolar mixture of the 7 proteins was digested, added to digested plasma, and then diluted with digested plasma to generate a 9-point response curve spanning the same concentration range (1–500 fmol/μL) in a digested-plasma background (1 μg/μL). Corresponding stable isotope–labeled internal standard (SIS) peptides were added to each sample at 50 fmol/μL [referred to as study II in (34 (link))]. We monitored 5 MRM transitions for each analyte and SIS peptide on a 5500 Q TRAP mass spectrometer (Applied Biosystems) by scheduled MRM with a target cycle time of 0.5 s, a retention-time window of 90 s, and an interscan delay of 3 ms. The 2 additional transitions were selected from previous optimization experiments conducted with the synthetic peptides. The pooled and filtered human plasma used for all experiments was from Bioreclamation.
For the 5-MRM data set, an equimolar mixture of the 7 proteins was digested, added to digested plasma, and then diluted with digested plasma to generate a 9-point response curve spanning the same concentration range (1–500 fmol/μL) in a digested-plasma background (1 μg/μL). Corresponding stable isotope–labeled internal standard (SIS) peptides were added to each sample at 50 fmol/μL [referred to as study II in (34 (link))]. We monitored 5 MRM transitions for each analyte and SIS peptide on a 5500 Q TRAP mass spectrometer (Applied Biosystems) by scheduled MRM with a target cycle time of 0.5 s, a retention-time window of 90 s, and an interscan delay of 3 ms. The 2 additional transitions were selected from previous optimization experiments conducted with the synthetic peptides. The pooled and filtered human plasma used for all experiments was from Bioreclamation.
