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.
Highlights
Discovery proteomics, relying on nonsupervised data dependent acquisition (DDA), is routinely used to effectively profile, with broad coverage, the proteome under investigation [1, 2]
Principle and Implementation of Internal Standard Triggered-Parallel Reaction Monitoring (IS-parallel reaction monitoring (PRM))—The control of the conditions and the reproducibility of peptide chromatographic separations is critical in targeted experiments
It allows narrowing down the elution time windows during which peptides are monitored in time-scheduled analyses with an immediate benefit on the number of time windows segmenting the full LC-MS/MS run, and on the total number of peptides that can be monitored per run under conditions favoring high-analytical performance
Summary
Sample Preparation— Generic Sets of 10 –20 SIL Peptides Supplemented with Landmark Peptides—Low-purity synthetic isotopically labeled peptides (PEPotecTM peptides), with C-terminal 15N and 13C-labeled arginine and lysine residues, were provided by Thermo Fisher Scientific (Ulm, Germany) and were prepared at a nominal concentration of 50 to 500 fmol/l in aqueous solution. For regular PRM analyses, the precursor ions selected for the pairs of SIL and endogenous peptides were targeted in Ϯ1 min elution time windows (based on spectral library and off-line recalibration using landmark peptides prior to analysis) using two variant acquisition methods. For the PRM and IS-PRM analyses of the mixture of 606 SIL peptides in a plasma sample, the quantification was performed for endogenous peptides systematically detected in triplicated analyses For these peptides and corresponding SIL peptides, the AUC of the six most intense fragment ions (as defined in spectral libraries) were calculated based on the co-elution profiles of the differentially labeled peptides and summed to obtain the AUC of the peptides. A tutorial describing the installation of the application and its usage is provided in supplementary Experimental Procedures
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