Effect of Collision Energy Optimization on the Measurement of Peptides by Selected Reaction Monitoring (SRM) Mass Spectrometry

Abstract

Proteomics experiments based on Selected Reaction Monitoring (SRM, also referred to as Multiple Reaction Monitoring or MRM) are being used to target large numbers of protein candidates in complex mixtures. At present, instrument parameters are often optimized for each peptide, a time and resource intensive process. Large SRM experiments are greatly facilitated by having the ability to predict MS instrument parameters that work well with the broad diversity of peptides they target. For this reason, we investigated the impact of using simple linear equations to predict the collision energy (CE) on peptide signal intensity and compared it with the empirical optimization of the CE for each peptide and transition individually. Using optimized linear equations, the difference between predicted and empirically derived CE values was found to be an average gain of only 7.8% of total peak area. We also found that existing commonly used linear equations fall short of their potential, and should be recalculated for each charge state and when introducing new instrument platforms. We provide a fully automated pipeline for calculating these equations and individually optimizing CE of each transition on SRM instruments from Agilent, Applied Biosystems, Thermo-Scientific and Waters in the open source Skyline software tool (http://proteome.gs.washington.edu/software/skyline).

Large-Scale Interlaboratory Study to Develop, Analytically Validate and Apply Highly Multiplexed, Quantitative Peptide Assays to Measure Cancer-Relevant Proteins in Plasma

“…The final MRM assay culminated in the three most abundant and interference-free transitions per peptide (transitions were selected for the unlabeled, 13 C/ 15 N-isotopically labeled, and U-15 N-isotopically labeled version of each peptide). Collision energy values used were based upon linear regression equations provided in Skyline for each vendor platform (40). The final list of MRM transitions for each platform is listed in Supplemental Table 2.…”

Analysis of host-cell proteins in biotherapeutic proteins by comprehensive online two-dimensional liquid chromatography/mass spectrometry

“…The ESI-MS conditions are as follows: electrospray capillary voltage 3.5 kV, cone voltage 35 V, source temperature 90°C, desolvation temperature 400°C and desolvation gas flow 800 L/hour. All MRM measurements were acquired at unit mass resolution (0.75 Da FWHM) for both MS1 and MS2 using dwell times ranging from 10-40 ms. Collision energies were assigned according to the formula CE = 0.034 x m/z + 3.3, 50 regardless of precursor charge state.…”

Cell-specific Labeling Enzymes for Analysis of Cell–Cell Communication in Continuous Co-culture

“…The TSQ Vantage was operated with a Q1 unit resolution of 0.7 full width at half-maximum, a Q3 of 0.7 full width at half-maximum, an ion spray voltage of 2200 V, and a capillary inlet temperature of 270°C. Peptide fragmentation was carried out in Q2 at 1.5 millitorr, and collision energies for each peptide were predicted (7). Each selected reaction monitoring (SRM) transition had a minimum dwell time of 20 ms, with cycle times of 1.2 s. The raw data files were produced in Xcalibur 2.1 (Thermo Scientific), and all data were processed using Skyline 2.1 (8).…”