The use of targeted proteomic experiments for the identification or isolation of a specific peptide from a complex mixture by ion-trap mass spectrometers is a battle-tested technique in use for the past several decades. The drawbacks of these experiments, mainly the limitations of the mass spectrometers themselves,1 have been slowly mitigated over the years by the advent of new processing power, faster scan times, and higher resolution mass spectrometers.1,2 Unfortunately, coinciding with these advances in technology, the complexity of the samples has also increased to include thousands of peptides or biomolecules. Moving into the fields of biomarker discovery and personalized medicine in the sense of clinical diagnosis, where the exact nature of the desired peptide may not be known due to upstream differential proteolysis of the product before analysis, the ideal setup would be a system that could monitor several ion transition states and use predictive fragmentation to assist in reanalysis of older samples. Ion-trap mass spectrometers offer a variety of advantages over quadrupole or Q-type instruments in the sense that multiple selected reaction monitoring (SRM) transitions can be obtained simultaneously without loss of processing power and a more reproducible fragmentation pattern for MS/MS analysis and additional identification techniques.
A large drawback of SRM assays is the revalidation of protein hits from previously run samples where there may not be additional material to analyze. In Hewel et al.,3 the authors report the ability to use targeted peptide monitoring (TPM) to show highly reproducible, quantitative and sensitive assays for the detection of heavy and light spiked samples of mouse cells in the identification of target products. Using an LTQ Orbitrap Velos mass spectrometer (Thermo Scientific), the SRM-style experiment was optimized. A total of 26 pairs labeled as peptides (light and heavy) were spiked into a biological sample from mouse cells. Using the retention times for the defined labeled peptides, the higher resolution nature of the Orbitrap allowed for the identification of low abundant peptide precursor ions that would normally be lost due to typical operating signal-to-noise ratios. The spiked peptides served as a marker for the instrument for the identification of the most abundant peptide hits within the retention time window established beforehand.
In conclusion, the LTQ Orbitrap Velos mass spectrometer provides a competent platform for the TPM assays due to high mass accuracy, high degree of calibration stability, excellent experiment-to-experiment comparison of fragmentation (and lab-to-lab fragmentation) and the scan time, and ability to muitiplex the ion acquisition.3 This allows for single-ion-based identification of products and the detection and reconstruction of lower abundant ions in complex mixtures. This report provides real life examples of the use of a high-resolution mass spectrometer for the identification of low abundance peptides in a TPM style assay.
1. Picotti, P. and Aebersold, B. (2012) ‘Selected reaction monitoring-based proteomics: workflows, potential, pitfalls and future directions‘, Nature Methods, 9 (6), (pp. 555-566)
2. Shi, T., et al., (2012) ‘Advancing the sensitivity of selected reaction monitoring-based targeted quantitative proteomics‘, Proteomics, 12 (8), (pp. 1074-1092)
3. Hewel, J.A., et al., (2012) ‘Targeted protein indentification, quantification and reporting for high-resolution nanoflow targeted peptdie monitoring‘, Journal of Proteomics, 12 (1), (pp. 1-14)