Gallien et al. (2015) provide a methodologically dense paper that sets out the protocols for internal standard triggered–parallel reaction monitoring (IS-PRM), a novel targeted data acquisition scheme for large-scale proteomics. In essence, the authors present a validated system for real-time instrument operation and data acquisition control, in addition to quantitative proteomics analysis for a larger target grouping. Ultimately, the research team posits that IS-PRM presents advantages over both serial reaction monitoring (SRM), and existing direct data acquisition (DDA) and targeted data acquisition (TDA) modes.
Using the drive toward biomarker discovery as an incentive where scientists analyze larger and larger peptide numbers under increasingly efficient instrument operation, Gallien et al. propose a targeted workflow with internal standard monitoring switching mass spectrometry operation between “watch mode” and “quantitative mode” for data acquisition. The team accomplished this with stable isotope labeled (SIL) internal peptide references spiked into experimental samples. In this way, they describe instrument control as dynamic and occurring in real time through continual monitoring of the tandem mass spectrometry (MS/MS) spectra generated during each experimental run.
The basis of the IS-PRM workflow is the use of SIL peptides to generate internal standards that are common across all experimental protein digests. The team employed various sources—including PEPotec and Pierce Peptide Retention Time Calibration Mix (both Thermo Scientific)—as internal standards to spike human plasma, urine or pooled HeLa cell peptide digests prior to liquid chromatography (LC)-MS/MS evaluation. In addition, the researchers also used a series of 13 SIL peptides as external references for instrument calibration between and within run times.
Gallien et al. used a number of chromatography systems and quadrupole-Orbitrap mass spectrometers during validation and operational analysis of the IS-PRM workflow. These included an UltiMate 3000 RSLCnano LC system in conjunction with Acclaim PepMap trap and analytical columns (all Thermo Scientific) for chromatographic separation. Following this, they analyzed the separated peptides on three different quadrupole-Orbitrap mass spectrometers—Q Exactive, Q Exactive Plus or Q Exactive HF mass spectrometers (all Thermo Scientific)—to generate spectral libraries. The team also used a triple quadrupole instrument, the TSQ Vantage mass spectrometer (Thermo Scientific) for SRM assay.
Once the team analyzed the spectral data with Xcalibur software v.2.2 and Pinpoint software v.1.3 (both Thermo Scientific), they also developed programming using C# for in-house scripts and an application programming interface (API) to automate data evaluation in real time.
Gallien et al. approached the study in four phases:
- Development and implementation phase
This phase included creation and optimization of MS operating parameters to set up the new data acquisition mode for dynamic instrument control during IS-PRM assays. The team set up monitoring window duration and MS acquisition time, and evaluating peptide numbers during monitoring. During this step, they assessed instrument operating conditions with the SIL peptide mixes, calibrating MS parameters for accurate and consistent internal and external reference standard handling.
- Implementation on quadrupole Orbitrap-based MS
During this stage, the researchers spiked human plasma with the SIL reference peptides, then defined the acquisition parameters that would ensure triggering the switch from “watching” to “quantitation” mode for IS-PRM. When they ran a “blank” sample, “quantitation” mode did not trigger, showing that instrument control did rely on SIL reference marker detection.
- Analytical performance
Using a dilution series, the team calculated limits of quantitation and detection for the analytes. They found these to be sufficiently sensitive and consistent for IS-PRM assay performance.
- Application to large-scale quantitative experiments
Following optimization and validation, the researchers switched to large-scale studies on the triple quadrupole instrument to evaluate performance under realistic experimental conditions. At this stage, Gallien et al. studied IS-PRM performance for various matrices, including pooled urine samples and a HeLa cell peptide digest. In this way, they could assess the effect of different matrices and larger numbers of peptides on assay performance.
In summary, the IS-PRM assay workflow quantified larger peptide numbers sensitively and consistently within a single experiment. Moreover, the workflow retained high analytical performance, maintaining consistency over time and across instruments through spectral library generation. Internal standards successfully drove real-time instrument operation, switching data acquisition modes between “watching” and “quantitative.”
Gallien et al. suggest that IS-PRM is an appropriate targeted data acquisition mode for large-scale screening as required for biomarker discovery.
1. Gallien, S., et al. (2015) “Large-scale targeted proteomics using internal standard triggered-parallel reaction monitoring,” Molecular and Cellular Proteomics, 14 (pp.1630-44). doi: 10.1074/mcp.O114.043968
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