MRM Quantitative Analysis
After synthesis and purification, each “heavy” idiotypic peptide is dissolved in a 50/50 acetonitrile/water buffer and sent for amino acid analysis to determine absolute concentration on the synthetic peptide stock solution. Each “heavy” peptide is then directly infused into the 4000 QTRAP mass spectrometer (MS) to determine the best collision energy for MS/MS fragmentation and two to four MRM transitions. Next, the neat “heavy” peptides are subjected to LCMS on the 4000 QTRAP to ensure peptide separation. The 4000 QTRAP instrument is run in the triple quadrupole mode, with Q1 set on the specific precursor m/z value (Q1 is not scanning), and Q3 set to the specific m/z value corresponding to a specific fragment of that peptide. In the MRM mode, a series of single reactions (precursor / fragment ion transitions where the collision energy is tuned to optimize the intensity of the fragment ions of interest) are measured sequentially, and the cycle (typically 1-2 sec) is looped throughout the entire time of the HPLC separation. MRM transitions are determined from the MS/MS spectra of the existing peptides. Typically, doubly charged precursors (or triply charged in some instances) are selected. Two transitions per peptide, corresponding to high intensity fragment ions, are then selected and the collision energy optimized to maximize signal strength of MRM transitions by using automation software. By using scheduled MRM (AB Analyst 1.5 software), where MRM transitions are based on retention time, we can effectively monitor about 500 peptides per run (~50 proteins), where each protein has 3 peptides and each peptide 3 MRMs along with a heavy labeled internal standard with the matching 3 MRMs. Data analysis on LC-MRM data is performed using AB Multiquant 1.1 software.