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 ((13)C and (15)N) 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 parallel reaction monitoring experiments. Detection and linearity of the instrument routinely spanned three orders of magnitude across the gradient (500 fmol to 0.5 fmol on column) and no systematic trend was observed for MMA of targeted peptides as a function of abundance by analysis of variance analysis (p = 0.17). Detection and linearity of the fifth isotopologue (i.e., 0.05 fmol on column) was dependent on the peptide and instrument scan type (MS1 vs PRM). We foresee that this standard will serve as a powerful method to conduct both intra-instrument performance monitoring/evaluation, technology development, and inter-instrument comparisons.

Quality control in mass spectrometry‐based proteomics

“…An important consideration when spiking synthetic peptides into other samples is that these peptides should not overlap with the original sample content. This can be avoided by using artificial, synthetically modified, peptides that are dissimilar from any naturally occurring peptides, or by isotopically labeling the synthetic peptides so that their mass is dissimilar from the mass of their naturally occurring peptide variants . These synthetic QC3 peptide mixtures are especially important to evaluate the performance of targeted approaches, such as selected reaction monitoring (SRM).…”

“…Instead, the reference RTs of the iRT peptides can be used to correct for variations in the RT of the other peptides detected in a single experiment or to align RTs across multiple experiments. Several QC standards containing iRT peptides have been proposed . These standards mostly vary slightly in the range of the LC gradient they can cover, but some standards have further advanced properties.…”

“…The dynamic range can be monitored if peptides are present in varying concentrations, as illustrated in Figure . QC samples can contain distinct peptides in different concentrations or isotopically labeled variants of the same peptide at different ratios . While the concentrations typically span two to four orders of magnitude, the ability to detect even the smallest concentrations indicates the capacity to detect low‐abundant peptides over the observed extensive proteome dynamic range .…”

“…This can be avoided by using artificial, synthetically modified, peptides that are dissimilar from any naturally occurring peptides, 46 or by isotopically labeling the synthetic peptides so that their mass is dissimilar from the mass of their naturally occurring peptide variants. [47][48][49] These synthetic QC3 peptide mixtures are especially important to evaluate the performance of targeted approaches, such as selected reaction monitoring (SRM). To be able to consistently monitor the transitions of specific peptides and to optimally schedule SRM experiments chromatographic stability is an essential prerequisite, which can be evaluated using these well-characterized peptides as their transitions should exhibit minimal run-to-run variation.…”

“…Several QC standards containing iRT peptides have been proposed. 49,86,116,119,120 These standards mostly vary slightly in the range of the LC gradient they can cover, but some standards have further advanced properties.…”