Mass spectrometry (MS) based proteomics have achieved a near-complete proteome coverage in humans and in several other organisms, producing a wealth of information stored in databases and bioinformatics resources. Recent implementation of selected/multiple reaction monitoring (SRM/MRM) technology in targeted proteomics introduced the possibility of quantitatively follow-up specific protein targets in a hypothesis-driven experiment. In contrast to immunoaffinity-based workflows typically used in biological and clinical research for protein quantification, SRM/MRM is characterized by high selectivity, large capacity for multiplexing (approx. 200 proteins per analysis) and rapid, cost-effective transition from assay development to deployment. The concept of SRM/MRM utilizes triple quadrupole (QqQ) mass analyzer to provide inherent reproducibility, unparalleled sensitivity and selectivity to efficiently differentiate isoforms, post-translational modifications and mutated forms of proteins. SRM-like targeted acquisitions such as parallel reaction monitoring (PRM) are pioneered on high resolution/accurate mass (HR/AM) platforms based on the quadrupole-orbitrap (Q-orbitrap) mass spectrometer. The expansion of HR/AM also caused development in data independent acquisition (DIA). This review presents a step-by-step tutorial on development of SRM/MRM protein assay intended for researchers without prior experience in proteomics. We discus practical aspects of SRM-based quantitative proteomics workflow, summarize milestones in basic biological and medical research as well as recent trends and emerging techniques.

• Klíčová slova
• Data independent acquisition (DIA), Parallel reaction monitoring (PRM), Selected/multiple reaction monitoring (SRM/MRM), Sequential windowed acquisition of all theoretical fragmentation spectra (SWATH), Tandem mass spectrometry (MS/MS), Targeted quantitative proteomics tutorial,
• MeSH
• hmotnostní spektrometrie * MeSH
• lidé MeSH
• posttranslační úpravy proteinů MeSH
• proteom MeSH
• proteomika metody   MeSH
• reprodukovatelnost výsledků MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• proteom MeSH

Targeted mass spectrometry-based proteomics approaches enable the simultaneous and reproducible quantification of multiple protein analytes across numerous conditions in biology and clinical studies. These approaches involve e.g. selected reaction monitoring (SRM) typically conducted on a triple quadrupole mass spectrometer, its high-resolution variant named pseudo-SRM (p-SRM), carried out in a quadrupole coupled with an TOF analyzer (qTOF), and "sequential window acquisition of all theoretical spectra" (SWATH). Here we compared these methods in terms of signal-to-noise ratio (S/N), coefficient of variance (CV), fold change (FC), limit of detection and quantitation (LOD, LOQ). We have shown the highest S/N for p-SRM mode, followed by SRM and SWATH, demonstrating a trade-off between sensitivity and level of multiplexing for SRM, p-SRM, and SWATH. SRM was more sensitive than p-SRM based on determining their LOD and LOQ. Although SWATH has the worst S/N, it enables peptide multiplexing with post-acquisition definition of the targets, leading to better proteome coverage. FC between breast tumors of different clinical-pathological characteristics were highly correlated (R2 >0.97) across three methods and consistent with the previous study on 96 tumor tissues. Our technical note presented here, therefore, confirmed that outputs of all the three methods were biologically relevant and highly applicable to cancer research.

• Klíčová slova
• Cancer, MRM/SRM, SWATH, Tissue, p-SRM,
• MeSH
• hmotnostní spektrometrie přístrojové vybavení   metody   MeSH
• lidé MeSH
• limita detekce MeSH
• nádory chemie   metabolismus   MeSH
• poměr signál - šum MeSH
• proteiny analýza   MeSH
• proteomika metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• proteiny MeSH