Reagent for Evaluating Liquid Chromatography–Tandem Mass Spectrometry (LC-MS/MS) Performance in Bottom-Up Proteomic Experiments

Abstract

We present a novel proteomic standard for assessing liquid chromatography–tandem mass spectrometry (LC-MS/MS) instrument performance, in terms of chromatographic reproducibility and dynamic range within a single LC-MS/MS injection. The peptide mixture standard consists of six peptides that were specifically synthesized to cover a wide range of hydrophobicities (grand average hydropathy (GRAVY) scores of −0.6 to 1.9). A combination of stable isotope labeled amino acids (13C and 15N) were inserted to create five isotopologues. By combining these isotopologues at different ratios, they span four orders of magnitude within each distinct peptide sequence. Each peptide, from lightest to heaviest, increases in abundance by a factor of 10. We evaluate several metrics on our quadrupole orbitrap instrument using the 6 × 5 LC-MS/MS reference mixture spiked into a complex lysate background as a function of dynamic range, including mass measurement accuracy (MMA) and the linear range of quantitation of MS1 and paralle...