Proteomics has moved from simple protein identification to targeted analysis that characterizes and quantifies tens to hundreds of peptides in a single run. High-resolution instruments such as the Q Exactive hybrid quadrupole-Orbitrap mass spectrometer (Thermo Scientific) can cope with this increase in data volume and analyte load, which is common in areas of research such as biomarker discovery, where scientists routinely hunt for targets amidst complex biological matrices. Ensuring accurate, efficient and robust proteomic analysis for a wide variety of low-abundance analytes across multiple samples is essential for early-stage investigation.
By generating high-resolution accurate-mass (HRAM) data, the Q Exactive mass spectrometer refines the initial discovery process, allowing researchers to drill down at an earlier stage to target candidates of interest for further study. A recent application note highlights the advantages this instrument brings to targeted protein quantitation, evaluating performance using a multiplexed selected ion monitoring (msx-tSIM) workflow in spiked samples of yeast whole-cell tryptic digest.1
Choosing the yeast whole-cell tryptic digest as an example of complex biological material, the scientists spiked increasing concentrations of heavy isotope-labeled peptide retention standards (Pierce Peptide Retention Time Calibration Mixture, Thermo Scientific) into the yeast digest. They used two concentrations of the yeast tryptic digest (5 ng/μL or 500 ng/μL) to recreate conditions where analytes would be present within low- or high-level backgrounds, respectively, of a complex biological matrix. Following liquid chromatography separation using an Acclaim PepMap C18 column (Thermo Scientific), the team extracted the retention times of the heavy peptide standards to set target selected ion monitoring (SIM) windows in the Q Exactive mass spectrometer.
The scientists acquired full scans and targeted msx-tSIM scans by isolating targets in the Q Exactive mass spectrometer C-trap before transferring the accumulated products to the Orbitrap mass analyzer for HRAM detection. They used this selective accumulation of ions within a narrow mass range to better target the analytes of interest, especially those in the preparations simulating low analyte abundance.
Comparing the scan data with those obtained via traditional selected reaction monitoring (SRM)-based assays, the scientists found that the Q Exactive mass spectrometer-based HRAM workflow with msx-tSIM scanning achieved lower limits of detection, faster processing, and better resolution. The benefits of these results are summarized as follows:
- The workflow avoids the need to create an SRM assay for quantification, a time-consuming extra step for analyte quantitation.
- The ability to specifically target ions in the quadrupole mass filter for SIM and MS/MS scans enriches analytes with low abundance, even in the presence of complex background materials.
- Faster scan speeds and multiplexed scanning function increases throughput and ability to handle complex samples. The msx-tSIM handled up to 10 targets in one scan.
- Higher-resolution scanning increases sensitivity for accurate identification and quantitation.
In conclusion, Zhang et al. describe the Q Exactive mass spectrometer workflow as suitable for discovery and targeted proteomics, facilitating both qualitative and quantitative research. They recommend the msx-tSIM strategy as an efficient and robust system for selecting strong candidates for further investigation in the early discovery phase of research.
Reference
1. Zhang, Y., et al. (2012) “HR/AM targeted peptide quantitation on a Q Exactive MS: A unique combination of high selectivity, sensitivity and throughput,” Application Note 554, Thermo Scientific.
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