Method of the Year
“nature methods” Method of the year 2012
Selected reaction monitoring mass spectrometry (SRM-MS) is a highly selected and sensitive method used to quantitate protein/peptide abundances in complex biological samples for targeted quantitation of proteins. SRM-MS is commonly utilised for small molecule analysis but has now developed as a mainstream technology for proteomics. SRM-MS assays are compatible with multiplexing, enabling the quantitation for 10s-100s of analytes in a single MS run. For absolute quantitative analysis, synthetic peptides containing a stable isotopically labelled amino acid are used as standards. This enables the performance of each assay to be assessed for limits of detection/quantitation and linearity. SRM-MS assays can also be used for relative quantitation of analytes across runs.
Nature Methods' choice for Method of the Year 2012 is targeted proteomics (SRM-MS). Historically, researchers seeking quantitative information on proteins have relied on antibodies or other affinity reagents for quantitation. However, antibodies vary greatly in their specificity and are unavailable for many proteins. Further, there is a general absence of robust antibodies to detect modified proteins such as specific phosphoproteins. APAF is a leader in the use and development of SRM-MS technology for the past 5 years. Both, Selective Reaction Monitoring (SRM) and High Resolution Multiple Reaction Monitoring (MRM-HR), are available at APAF. The advantage of these techniques is that you don’t require antibodies and you can multiplex detection of many proteins in the one run.
The diagram below shows a comparison of conventional data-dependent analysis to targeted MRM-MS on a triple quadrupole mass spectrometer.
In data dependent MS/MS (a) the mass spectrometer is attempting to identify all fragment ions and you get a full scan MS/MS spectrum. In targeted MS/MS or MRM-MS (b) what you are looking for is predefined and fragment ion detection is based on the user-specific list.
What does this mean for you the scientist? A more targeted approach can offer the following;
- Validation of protein biomarkers – Discovery to verification either based on previous discovery work or theoretical
- Antibodies have a limited life and can get contaminated so buying more can be expensive.