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SRM/MRM

Liquid Chormatography-Selected Reaction Monitoring/Mass Spectrometry (LC-SRM/MS) is a technique commonly associated with the selective and sensitive detection of small molecules (<1000Da). The technique is commonplace in the clinical, pharmaceutical and environmental monitoring fields and is recognized for enabling robust, high throughput assays. LC-SRM/MS is an emerging technology for the quantitation of proteins. The principle underlying this approach is one of surrogacy; a proteolytic peptide produced from the enzymatic digestion of a protein can be monitored and quantified using LC-SRM/MS. The calculated molar amount of the surrogate peptide can be used to infer the concentration of the protein, assuming a 1:1 mole ratio between the peptide and the protein. This method offers an opportunity to dramatically reduce assay development times for proteins of interest and has all of the positive attributes associated with small molecule applications.

This service is designed to enable access to this emerging technology for use on your own biological systems. This service involves the quantitation of ten proteins in a ten samples using single point calibration. Internal standard peptides are not included in the service but MS Bioworks is happy to assist in sourcing and purchasing suitable reagents.

The steps involved in a Selected Reaction Monitoring experiment are:

• Method development – Peptide selection via a combined in-silico and empirical approach using Skyline software. We aim to select five surrogate peptides for each protein in the assay but this number is protein specific.
• Sample digestion – robotic reduction and alkylation followed by trypsin digestion.
• Optional – addition of synthetic peptide standards.
• LC/MS/MS – Robust liquid chromatography to yield high sensitivity; interfaced to a TSQ Quantum Ultra to allow sensitive time scheduled data acquisition.
• Data analysis – Data are analyzed using Skyline software.
• Report generation – PDF report, Excel and Scaffold files. Data are rationalized and presented to ensure clarity and the ability to understand the results.

Key features of a Selected Reaction Monitoring experiment are:

• Protein quantitation without affinity reagents.
• Readily multiplexed
• Use of stable label peptides enables reporting of absolute protein concentrations