The NIMML Proteomics team provides NIMML’s proteomics needs. The Proteomics service in NIMML is administered by Caprion Proteomics Inc. Caprion is the leading provider of proteomics based services to the life science industry. The company has established for over a decade a proteomics platform that has integrated state of the art mass spectrometry with deep expertise in biological sample preparation as well as sophisticated large scale data analysis.
The NIMML team has developed and deployed multiplex targeted proteomics analyses to measure the expression changes of the protein products of genes of interest that were identified by the program’s transcriptomic and immunologic analyses. The targeted proteomics analyses are conducted using Multiple Reaction Monitoring (MRM) Mass Spectrometry, a method that specifically and sensitively detects the selected peptides.
Multiple Reaction Monitoring Assays
Candidate protein biomarkers can be selected from the literature or by genomics and proteomics discovery efforts. The MRM method employs liquid chromatography coupled to triple quadrupole mass spectrometers. Each quadrupole can be set to filter selected peptide ions. For an MRM assay, a specific peptide that corresponds to a protein of interest is selected by the first quadrupole. The peptide is then fragmented in the second quadrupole. Subsequently, a filter is applied in the third quadrupole to allow only one specific fragment, also referred to as a transition, to reach the detector. Typically, two peptides per protein are monitored, and two transitions per peptide are measured. Because of the double selection approach, which improves signal-to-noise and reduces interference, and multiple measurements per protein, MRM is one of the most specific assays for protein measurement. In addition, current instrumentation allows for the measurement of hundreds of proteins in a single sample, making MRM an ideal assay to verify and validate candidate biomarkers or perform high-throughput measurements of a panel of markers.
To further increase sensitivity, MRM can also be used as a readout for samples prepared by immunoaffinity enrichment of target proteins. Antibodies that display cross-reactivity can be used in such assays because the specificity is assured by the MRM assay. Such antibody-assisted MRM assays are highly linear and, when applied against plasma proteins, typically show lower limits of quantification in the low nanogram per ml range.
Targeted mass spectrometry approaches for protein biomarker verification. Meng Z, Veenstra TD. J Proteomics. 2011 Apr 21.
Multiple reaction monitoring for quantitative biomarker analysis in proteomics and metabolomics. Kitteringham NR, Jenkins RE, Lane CS, Elliott VL, Park BK. J Chromatogr B Analyt Technol Biomed Life Sci. 2009 May 1;877(13):1229-39.
Protein biomarker quantification by mass spectrometry. Hunter J, Paramithiotis E. Expert Opin Med Diagn. 2009;3:1