Large-Scale Targeted Proteomics Using Internal Standard Triggered-Parallel Reaction Monitoring (IS-PRM)

Abstract

Targeted high-resolution and accurate mass analyses performed on fast sequencing mass spectrometers have opened new avenues for quantitative proteomics. More specifically, parallel reaction monitoring (PRM) implemented on quadrupole-orbitrap instruments exhibits exquisite selectivity to discriminate interferences from analytes. Furthermore, the instrument trapping capability enhances the sensitivity of the measurements. The PRM technique, applied to the analysis of limited peptide sets (typically 50 peptides or less) in a complex matrix, resulted in an improved detection and quantification performance as compared with the reference method of selected reaction monitoring performed on triple quadrupole instruments. However, the implementation of PRM for the analysis of large peptide numbers requires the adjustment of mass spectrometry acquisition parameters, which affects dramatically the quality of the generated data, and thus the overall output of an experiment. A newly designed data acquisition scheme enabled the analysis of moderate-to-large peptide numbers while retaining a high performance level. This new method, called internal standard triggered-parallel reaction monitoring (IS-PRM), relies on added internal standards and the on-the-fly adjustment of acquisition parameters to drive in real-time measurement of endogenous peptides. The acquisition time management was designed to maximize the effective time devoted to measure the analytes in a time-scheduled targeted experiment. The data acquisition scheme alternates between two PRM modes: a fast low-resolution "watch mode" and a "quantitative mode" using optimized parameters ensuring data quality. The IS-PRM method exhibited a highly effective use of the instrument time. Applied to the analysis of large peptide sets (up to 600) in complex samples, the method showed an unprecedented combination of scale and analytical performance, with limits of quantification in the low amol range. The successful analysis of various types of biological samples augurs a broad applicability of the method, which is likely to benefit a wide range of proteomics experiments.

Fig. 1.

Acquisition efficiency of time-scheduled PRM methods. A, Comparison of peptide chromatographic peak width (typically 30 s) with elution time monitoring windows employed in current targeted acquisition methods used in proteomics (typically 3 min). B, Model representing the MS acquisition time devoted to the sequential PRM measurement of 12 pairs of SIL and endogenous (“Heavy”/“Light”) peptides on a Q-Exactive instrument at a given chromatographic time using a transient length of 128 ms (resolution of 35,000 (at m/z 200)) and a synchronized maximum fill time of 110 ms, resulting in a cycle time of 3.1 s. The mismatch between scheduled monitoring window and peptide chromatographic width translated in an acquisition efficiency of 15%. This corresponded to a number of four out of the 24 peptides measured per cycle when they were actually eluting.

Fig. 2.

Data acquisition scheme of internal standard - triggered parallel reaction monitoring method. A, The instrument operated in two alternating PRM modes: the watch mode and the quantitative mode. In the watch mode, only SIL peptides (internal standards) were continuously monitored in their 1-min dynamic monitoring window (based on external landmark peptides). The detection of a SIL peptide (e.g. DADPDTFFAK in this example) was performed through on-the-fly extraction and evaluation of the MS/MS data acquired in watch mode by spectral matching against a library of reference MS/MS spectra. This detection triggered a switch from the watch mode (stopped for the given peptide) to the quantitative mode to measure the corresponding pair of SIL and endogenous peptide for a predefined monitoring window matching the peptide chromatographic peak width (typically 0.5 min). B, The acquisition parameters used in watch mode favored speed over data quality. In the model, a transient length of 64 ms (resolution of 17,500 (at m/z 200) on Q-Exactive instrument) and a synchronized maximum fill time of 60 ms were used. By contrast, the acquisition parameters used in quantitative mode, that is, a transient length of 256 ms (resolution of 70,000 (at m/z 200) on a Q-Exactive instrument) and a synchronized maximum fill time of 250 ms, enhanced data quality. In the model, such parameters enabled the PRM measurement of 15 peptides per cycle (for a cycle time of 3.5 s), including six pairs of SIL and endogenous peptides at the time of their actual elution. This resulted in an acquisition efficiency of 90% (overhead time of 200 ms estimated per cycle). An increase in the dynamic monitoring window of SIL peptides in watch mode to 2 min moderately affects the acquisition efficiency. Under this condition and keeping unchanged the other parameters in the model, nine PRM scans would be acquired in watch mode, resulting in an acquisition efficiency of 80%.

Fig. 3.

Comparison of cycle times in PRM and IS-PRM analyses of 93 pairs of SIL and endogenous peptides in a plasma sample. The cycle times observed over the entire analyses were plotted together with the corresponding number of peptides monitored in the regular PRM analysis. The average cycle time values between 15 and 52 min, corresponding to the elution range of 90 of the pairs of peptides were also displayed, corresponding to 0.9 and 1.2 s for PRM and IS-PRM, respectively. The acquisition parameters used in each method to measure SIL and endogenous peptides on a Q-Exactive Plus instrument were detailed.

Fig. 4.

Comparison of the quantification performance of SRM, regular PRM and IS-PRM analyses of a dilution series of 93 SIL peptides spiked in a plasma sample (389 fragment ions common to the various methods evaluated). The number of transitions that could be used for reliable quantification at the different dilution points was indicated.

Fig. 5.

Comparison of the quality of data generated by SRM, regular PRM, and IS-PRM analyses of a dilution series of 93 SIL peptides spiked in a plasma sample. The transitions (SRM) or fragment ion traces (PRM and IS-PRM) extracted from the measurement by each technique of the SIL peptide DGAGDVAFVK (m/z 493.755, 2⁺), representing human serotransferrin, spiked at 150 amol/μl in a plasma sample were displayed. The fragment ion signals acquired in IS-PRM analysis exhibited co-eluting profiles and high signal to noise ratios whereas those acquired in regular PRM analysis showed limited ion statistics and less consistent relative intensities. Most of the SRM transitions were heavily interfered with background signals, leading to an undistinguishable elution profile.

Fig. 6.

Comparison of the detection and quantification performance obtained by triplicated IS-PRM and regular PRM (PRM-method A and PRM-method B) analyses of 606 pairs of SIL and endogenous peptides in a plasma sample. The fragment ion traces were extracted for the measurement of the endogenous peptide LTVGAAQVPAQLLVGALR (m/z 894.042, 2⁺), surrogate of human monocyte differentiation antigen CD14, by each method (acquisition parameters displayed in Table I). The peptide was systematically detected in triplicated analyses using IS-PRM and PRM-method A but did not satisfy the acceptance criteria using PRM-method B because of the low signal-to-noise ratio or the nondetection of its fragment ions. A cycle time of 1.9 s was observed in triplicated IS-PRM analyses, ensuring precise quantification results (CV of 5%) whereas acquisition parameters used in PRM-method A resulted in a significant increase in cycle time (6.3 s) and in limited precision (CV of 23% in triplicated analyses).