Parallel reaction monitoring for high resolution and high mass accuracy quantitative, targeted proteomics

Abstract

Selected reaction monitoring on a triple quadrupole mass spectrometer is currently experiencing a renaissance within the proteomics community for its, as yet, unparalleled ability to characterize and quantify a set of proteins reproducibly, completely, and with high sensitivity. Given the immense benefit that high resolution and accurate mass instruments have brought to the discovery proteomics field, we wondered if highly accurate mass measurement capabilities could be leveraged to provide benefits in the targeted proteomics domain as well. Here, we propose a new targeted proteomics paradigm centered on the use of next generation, quadrupole-equipped high resolution and accurate mass instruments: parallel reaction monitoring (PRM). In PRM, the third quadrupole of a triple quadrupole is substituted with a high resolution and accurate mass mass analyzer to permit the parallel detection of all target product ions in one, concerted high resolution mass analysis. We detail the analytical performance of the PRM method, using a quadrupole-equipped bench-top Orbitrap MS, and draw a performance comparison to selected reaction monitoring in terms of run-to-run reproducibility, dynamic range, and measurement accuracy. In addition to requiring minimal upfront method development and facilitating automated data analysis, PRM yielded quantitative data over a wider dynamic range than selected reaction monitoring in the presence of a yeast background matrix because of PRM's high selectivity in the mass-to-charge domain. With achievable linearity over the quantifiable dynamic range found to be statistically equal between the two methods, our investigation suggests that PRM will be a promising new addition to the quantitative proteomics toolbox.

Fig. 1.

Schematic representation of SRM (A) and PRM (B) as performed on QqQ and QqOrbi (or QqTOF) instrumentation, respectively. In SRM (A), each product ion transition (white circle) is serially monitored (from 1 to 5) one at a time in distinct scans. In PRM (B), all product ion transitions (1–5, and all possible product ions, shown as black circles) are analyzed/monitored in one concerted, high resolution and high mass accuracy mass analysis. Q1 and Q3 refer to the first and third mass-resolving quadrupoles of the QqOrbi (Q1 only) and QqQ, and q2 to the quadrupole (or cell, in the QqOrbi case) in which beam-type CAD is performed. The isolation widths employed for each device in both experimental and theoretical data are given below each respective device. C, Theoretical comparison of the rate of correctly identifying a target peptide (as true positive rate in percent, TPR) from all theoretically possible peptides in the human tryptic peptidome in SIM and reaction monitoring experiments in which 1, 2, and 3 y-ion transitions (labeled as 1T, 2T, and 3T, respectively) are monitored for Orbitrap or TOF instruments (±5 ppm) and QqQ (±250 ppm) for the 25 peptides used in this study in their light and heavy forms (50 total peptides). Count refers to the average number of possible confounding species, including the target peptide, represented by the boxplots. TPR is calculated as 100/count.

Fig. 2.

Extracted PRM score chromatograms (XSC, ±5 ppm, gray) overlaid with XICs (±5 ppm, blue; 7-point boxcar smoothed) and single-scan PRM spectra for peptide AETLVQAr (#17) isolated at ±1 Th under neat (left) and matrix-containing (right) conditions from 2 pm to 200 nm. Red dotted line in XSC plots represents the score acceptance threshold (8) for this peptide. Product ions detected in each spectrum are highlighted and the spectral score is labeled. Yellow arrows in each XIC/XSC plot indicate the retention time at which the associated single-scan spectrum was acquired. Hashed area in XIC/XSC plots designates the retention time period during which peak elution was expected based on the ±3σ range around the average retention time observed in 200 nm analyses.

Fig. 3.

A, Comparison of QqOrbi PRM detection at ±1 Th with QqQ SRM for peptide GVSAFSTWEk (#1) at 200 pm and 2 nm in the presence of matrix. Transition XICs are shown for the entire duration over which the peptide was targeted, with a zoom-in on the relevant time period in the QqQ SRM case (from the region shaded in gray in the chromatograms at the far right). Additional y-type product ions present in the PRM data are shown in gray. XICs were extracted at ±5 and ±250 ppm for PRM and SRM, respectively. The maximum spectral score attained at each concentration in PRM is also labeled. B, Lowest concentration detected (as number of attomoles of peptide on column) for each peptide in neat and matrix-containing experiments for the 14 peptides targeted in both SRM and PRM.

Fig. 4.

Comparison of linearity as %RSD and adjusted %RSD (supplemental Equation S1) for the 14 shared peptides targeted in QqOrbi PRM experiments (y axis) and QqQ SRM (x axis) under neat and matrix-containing conditions. Solid horizontal lines and dotted vertical lines represent the mean %RSD value, also labeled in the plot, of the associated data set. Data falling in the gray region demonstrate greater linearity in PRM experiments. Data in the white region demonstrate greater linearity in SRM experiments.