Selected reaction monitoring (SRM), also called multiple reaction monitoring, has become an invaluable tool for targeted quantitative proteomic analyses, but its application can be compromised by nonoptimal selection of transitions. In particular, complex backgrounds may cause ambiguities in SRM measurement results because peptides with interfering transitions similar to those of the target peptide may be present in the sample. Here, we developed a computer program, the SRMCollider, that calculates nonredundant theoretical SRM assays, also known as unique ion signatures (UIS), for a given proteomic background. We show theoretically that UIS of three transitions suffice to conclusively identify 90% of all yeast peptides and 85% of all human peptides. Using predicted retention times, the SRMCollider also simulates time-scheduled SRM acquisition, which reduces the number of interferences to consider and leads to fewer transitions necessary to construct an assay. By integrating experimental fragment ion intensities from large scale proteome synthesis efforts (SRMAtlas) with the information content-based UIS, we combine two orthogonal approaches to create high quality SRM assays ready to be deployed. We provide a user friendly, open source implementation of an algorithm to calculate UIS of any order that can be accessed online at http://www.srmcollider.org to find interfering transitions. Finally, our tool can also simulate the specificity of novel data-independent MS acquisition methods in Q1-Q3 space. This allows us to predict parameters for these methods that deliver a specificity comparable with that of SRM. Using SRM interference information in addition to other sources of information can increase the confidence in an SRM measurement. We expect that the consideration of information content will become a standard step in SRM assay design and analysis, facilitated by the SRMCollider.

• PMID: 22535207
• DOI full text
• PMC full text
• PubMed
• PubTator

Reference Type

Journal Article

Authors

Röst H, Malmström L, Aebersold R

Primary Lit For

Additional Lit For

Review For