Selected reaction monitoring for quantitative proteomics: a tutorial

By V. Lange, Paola Picotti, B. Domon, R. Aebersold. Molecular Systems Biology, 2008

1,417 citations recorded by the source corpus.

Systems biology relies on data sets in which the same group of proteins is consistently identified and precisely quantified across multiple samples, a requirement that is only partially achieved by current proteomics approaches. Selected reaction monitoring (SRM)—also called multiple reaction monitoring—is emerging as a technology that ideally complements the discovery capabilities of shotgun strategies by its unique potential for reliable quantification of analytes of low abundance in complex mixtures. In an SRM experiment, a predefined precursor ion and one of its fragments are selected by the two mass filters of a triple quadrupole instrument and monitored over time for precise quantification. A series of transitions (precursor/fragment ion pairs) in combination with the retention time of the targeted peptide can constitute a definitive assay. Typically, a large number of peptides are quantified during a single LC‐MS experiment. This tutorial explains the application of SRM for quantitative proteomics, including the selection of proteotypic peptides and the optimization and validation of transitions. Furthermore, normalization and various factors affecting sensitivity and accuracy are discussed.

Subfields: Molecular Biology, Spectroscopy

View Paper: https://www.semanticscholar.org/paper/c1fef3c5743c8161bc32526849e61c367c2d65bd

How researchers see this paper

The researchers whose trusted networks rank it highest, strongest advocate first. Every researcher gets a different order.

In Molecular Biology

In Spectroscopy

Citations in the corpus

Applying mass spectrometry-based proteomics to genetics, genomics and network biology (2009, background): “More recently, targeted proteomics workflows have been introduced to overcome these shortcomings of shotgun MS proteomic”
The Skyline Ecosystem: Informatics for Quantitative Mass Spectrometry Proteomics (2017, background): “Other Skyline Peptide Settings that affect results of peptide list generation are common biochemical sample preparation concerns such as missed cleavages, oxidized methionine, and peptide amino acid length (Anderson and Hunter 2005; Lange et al. 2008; Prakash et al. 2009).”
Less label, more free: Approaches in label‐free quantitative mass spectrometry (2011, background): “Additionally, the acquisition of reproducible quantitative data points over the chromatographic elution for any given peptide depends upon the balance between the number of data points accumulated per transition and the total number of transitions analysed in each dwell time [114].”
A survey of computational methods and error rate estimation procedures for peptide and protein identification in shotgun proteomics (2010, methodology): “alternative strategies such targeted protein identification using SRM [239].”
Parallel Reaction Monitoring for High Resolution and High Mass Accuracy Quantitative, Targeted Proteomics* (2012, background): “SRM achieves the reproducibility and sensitivity that the shotgun approach lacks, and even offers a route to determine absolute abundance (21).”
Data‐independent acquisition‐based SWATH‐MS for quantitative proteomics: a tutorial (2018, positive): “Generally, around 10 acquired data points are considered necessary to reconstruct chromatographic peak shapes and to perform accurate quantification (Fig 3C) (Matthews & Hayes, 1976; Lange et al , 2008).”
Targeted Data Extraction of the MS/MS Spectra Generated by Data-independent Acquisition: A New Concept for Consistent and Accurate Proteome Analysis* (2012, background): “…detection of the targeted precursors but proceeds by the acquisition, sequentially across the LC retention time domain, of predefined pairs of precursor and product ion masses, called transitions, several of which constitute a definitive assay for the detection of a peptide in a complex sample (8).”
Plasma Proteome Database as a resource for proteomics research: 2014 update (2013, background): “Proteotypic peptides that uniquely represent a protein are first selected, and assays are developed, which take into account the retention time of the peptides, their m/z values and measurement of specific fragment ions (39).”