Multiplexed, Scheduled, High-Resolution Parallel Reaction Monitoring on a Full Scan QqTOF Instrument with Integrated Data-Dependent and Targeted Mass Spectrometric Workflows

Abstract

Recent advances in commercial mass spectrometers with higher resolving power and faster scanning capabilities have expanded their functionality beyond traditional data-dependent acquisition (DDA) to targeted proteomics with higher precision and multiplexing. Using an orthogonal quadrupole time-of flight (QqTOF) LC-MS system, we investigated the feasibility of implementing large-scale targeted quantitative assays using scheduled, high resolution multiple reaction monitoring (sMRM-HR), also referred to as parallel reaction monitoring (sPRM). We assessed the selectivity and reproducibility of PRM, also referred to as parallel reaction monitoring, by measuring standard peptide concentration curves and system suitability assays. By evaluating up to 500 peptides in a single assay, the robustness and accuracy of PRM assays were compared to traditional SRM workflows on triple quadrupole instruments. The high resolution and high mass accuracy of the full scan MS/MS spectra resulted in sufficient selectivity to monitor 6-10 MS/MS fragment ions per target precursor, providing flexibility in postacquisition assay refinement and optimization. The general applicability of the sPRM workflow was assessed in complex biological samples by first targeting 532 peptide precursor ions in a yeast lysate, and then 466 peptide precursors from a previously generated candidate list of differentially expressed proteins in whole cell lysates from E. coli. Lastly, we found that sPRM assays could be rapidly and efficiently developed in Skyline from DDA libraries when acquired on the same QqTOF platform, greatly facilitating their successful implementation. These results establish a robust sPRM workflow on a QqTOF platform to rapidly transition from discovery analysis to highly multiplexed, targeted peptide quantitation.

Figure 1

Standard concentration curves for stable isotope-labeled and acetyllysine-containing peptides. (A) Stable isotope-labeled acetylated peptide LVSSVSDLPKacR* (HMGCS2) was monitored spanning a concentration range from 63 attomol/μL to 25 femtomol/μL (m/z 626.86²⁺), acquiring three replicates (1 μL sample on column). Peptides were spiked into a simple matrix containing a six digested protein mix at 25 fmol/μL each, and acquired on a TripleTOF 5600 mass spectrometer (QqTOF) in PRM mode in triplicate. Extracted peak areas for the top 5 MS/MS fragment ions were summed per peptide. (B) Peak area CVs across three replicates for each concentration point (0.063–25 fmol/μL) are shown for six targeted acetyllysine peptides with 20% CV cutoff indicated (Table S-1B, Supporting Information); K* = ¹³C₆¹⁵N₂-Lys and R* = ¹³C₆¹⁵N₄-Arg.

Figure 2

Response curve for a digested 6 protein mix spiked into a complex matrix (C. elegans whole cell lysate). (A) The mixture of six predigested proteins was spiked into digested C. elegans whole cell lysate (1 μg/μL and 1 μg on column) at 8 concentrations from 15 attomol/μL to 62.5 femtomol/μL (0 = blank, 0.015–62.5 fmol/μL, loading 1 μL sample on column). Two replicate concentration curves were acquired on the TripleTOF 5600 (QqTOF in PRM mode, top) and on the QTRAP 5500 (QQQ in SRM mode, bottom), respectively. Peak areas of extracted fragment ion transitions are summed per peptide. Curves are displayed for peptides HLVDEPQNLIK²⁺ (m/z 653.36) and YSTDVSVDEVK²⁺ (m/z 621.30), respectively. (B) Peak area CV display for triplicate acquisitions of a digested 6 protein mix (50 fmol/μL) spiked into a complex C. elegans matrix (1 μg/μL) monitoring 15 peptides in PRM mode with 10–24 transitions per peptide (50 fmol analyte injected). Each extracted fragment ion transition is displayed as a vertical colored line. 216 of 240 transitions showed CVs < 20% (Table S-2F, Supporting Information).

Figure 3

Workflow for highly multiplexed, RT scheduled sPRM assay development. (A) Spectral libraries were built from database searches from data-dependent acquisitions of a yeast extract. DDA files were imported into Skyline, and RTs are determined using MS1 Filtering for peptides to be targeted in the PRM assay. Skyline generates an instrument method with 2 min scheduling windows per peptide. sPRM acquisitions were imported into Skyline extracting fragment ion peak areas. (B) Extracted ion chromatogram of PRM fragment ions for DPIGITTLYMGR, spectral library MS/MS, and the corresponding peak area replicate view displaying the theoretical fragment ion distribution as simulated from the spectral library, and fragment ion XICs from one targeted sPRM acquisition. (C, D) Acquisition of three sPRM replicates monitoring >500 peptides from yeast lysate: (C) Peak area CVs from 3830 individual fragment ions when extracting peak areas from up to 10 fragment ions from each of the 503 precursor ions monitored. (D) Peak area CVs for the sum of the top 5 fragment ions from each of 503 precursor ions (Table S-3A-B, Supporting Information).