ProteinQuantSRM-Dx provides SRM (aka MRM) method design service to the bioanalytical community. The company was founded in 2023 by Bob Xiong in North Carolina. Bob has 15 years of industry experience in protein quantification by mass spectrometry. Prior to establishing ProteinQuantSRM-Dx, Bob held leadership positions in targeted proteomics at Joinn Laboratories, ICON, Covance, Labcorp, and Tandem Labs.
What We Do
SRM is widely accepted as the method of choice for protein quantification in biological samples. However, SRM is inherently burdened by the potential risk of co-eluting and indistinguishable precursor and product ions derived from the background proteome. The scientific community has duly recognized the challenge associated with the ion redundancy interference in SRM assays (Sherman, J., et al. 2009 How specific is my SRM?: The issue of precursor and produ t ion redundancy, Proteomics, 9(5): 1120-1123; Rost, H., et al. 2012 A computational tool to detect and avoid redundancy in selected reaction monitoring, Mol. Cell. Proteomics, 11(8): 540-549). At ProteinQuantSRM-Dx, we focus on in silico evaluation of Q1/Q3 transitions against the background proteome (e.g. human, monkey, mouse, yeast, fruit fly, Arabidopsis, etc.) to identify redundant ions that might interfere with the SRM assay specificity. Our goal is to aid our clients in avoiding redundant transitions at the outset of SRM method development.
Experimental data presented at 2013 ASMS by Xiong, B., et al.
Example Of Ion Redundancy (Tryptic Peptides In Human Serum)
The example below shows two tryptic peptides derived from two human proteins, respectively. Both peptides exhibit the same retention time at 2.90 minutes under the tested reversed phase LC-MS/MS conditions (column: Agilent Poroshell 120 EC-C18, 2.7 um, 2.1x50mm, MPA: 95/5 water/acetonitrile with 0.1% formic acid: MPB: 50/50 methanol/acetonitrile with 0.1% formic acid, gradient: 0%-50%B (7 minutes) -> 50%-100%B (0.1 minute) -> 100%B (0.5 minute) -> 100%-0%B (0.1 minute) -> 0%B (0.8 minute), instrument: SCIEX API 5000). The precursor ions for the two peptides are indistinguishable (doubly charged m/z 468.2) on a triple quadrupole mass spectrometer. The y6 product ions for the two peptides are also indistinguishable (singly charged m/z 708.5). It is clear that the observed single peak in human serum spiked with both proteins P19447 and P59044 represents two sources, i.e. the peak area in the two-protein spiked sample (sample 4) is roughly the sum of peak areas in the single-protein spiked samples (sample 2 and sample 3).
Synthetic SIL Internal Std Peptide (winged, before digestion)
Peak Area (sample 4) = Peak Area (sample 2) + Peak Area (sample 3)
Tryptic pepitdesINTIFISKandVQTLFLSKin sample 4 cannot be distinguished due to ion redundancy interference. (i.e. the two peptides contributed to forming a single peak)
