Selective Reaction Monitoring for Quantitation of Cellular Proteins
Proteins and proteomes are dynamic and complex. The accurate identification and measurement of their properties such as abundance, location, and turnover are challenging tasks. Even though high-throughput proteomics has significantly evolved, the techniqu
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Introduction Proteomics has evolved progressively and currently allows identification and quantification of large sets of proteins with good precision and across several orders of magnitude. This process reached its highpoint recently with the publication of the draft of human proteome [1, 2]. However, the proteome is dynamic as it changes constantly and it is significantly different in both composition and expression levels of individual components across cells, tissues, organs, and individuals. Because of this, the capabilities of high-throughput proteomic methods based on liquid chromatography tandem mass spectrometry (LC-MS/MS) to analyze multiple samples in a timely and reproducible manner are still considered to be limited [3]. In order to address this, targeted proteomics approaches have now focused on precise quantitation of specific sets of proteins in multiple samples or time points. In this way, the monitoring of dynamic proteome changes has emerged as a complementary strategy to complete and validate proteomic and transcriptomic datasets.
Paul C. Guest (ed.), Multiplex Biomarker Techniques: Methods and Applications, Methods in Molecular Biology, vol. 1546, DOI 10.1007/978-1-4939-6730-8_18, © Springer Science+Business Media LLC 2017
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Vitor M. Faça
The mass spectrometry-based strategy termed selected reaction monitoring (SRM), also known as multiple reaction monitoring (MRM), has been applied frequently for accurate quantitation of small molecules such as drugs or metabolites, and it is now being applied progressively more to the study of peptides and proteins [4]. This increasing interest in options for direct quantitation of proteins or peptides is also driven by limitations and lack of reproducibility of the antibody-based methods [5]. However, it should be stated that all methods have their limitations. The SRM method is mainly based on selecting and quantifying a specific set of peptides (proteotypic peptides) derived from a target list of proteins of interest using LC-MS/ MS analysis [6]. SRM takes advantage of the ion filtering capabilities of tandem quadrupole-based equipment, allowing selection of precursors and respective fragment ions produced by CID (collision-induced dissociation) to detect low abundance species among complex mixtures and across four to five orders of magnitude. Due to the rapid tandem quadrupole duty cycle that occurs over milliseconds and the online chromatographic separation of peptides, numerous precursor–fragment ion pairs known as SRM transitions can be monitored simultaneously. This facilitates quantitative experiments involving several different samples in a timely manner. Since SRM methods efficiently quantitate peptides, the accurate monitoring of proteomic changes in complex samples requires the inclusion of reproducible and efficient enzymatic digestion of proteins in the method. Also, the peptides selected as indicators of protein abundance must meet several characteristics, including the possibility of chemical synthesis to generate these as stand
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