Selected reaction monitoring–based proteomics: workflows, potential, pitfalls and future directions

Picotti, Paola; Aebersold, Ruedi

doi:10.1038/nmeth.2015

Cited by 1,163 publications

(955 citation statements)

References 115 publications

Supporting

Mentioning

926

Contrasting

Unclassified

Order By: Relevance

“…SRM relies on a three‐step process, the selection of precursor ions in a tandem mass spectrometer, the fragmentation of these ions in a collision chamber, and the monitoring of selected fragment ions, termed SRM transitions (Picotti & Aebersold, 2012). As an important preparatory step, the SRM workflow requires the identification of peptides, so‐called proteotypic peptides (PTPs), that are distinctive for a given protein and display high ionization efficiency, and the validation of optimized transitions specific for each PTP (Picotti & Aebersold, 2012).…”

Section: Resultsmentioning

confidence: 99%

“…As an important preparatory step, the SRM workflow requires the identification of peptides, so‐called proteotypic peptides (PTPs), that are distinctive for a given protein and display high ionization efficiency, and the validation of optimized transitions specific for each PTP (Picotti & Aebersold, 2012). In pilot experiments, we identified the two most suited PTPs for each centrosomal protein to be quantified (Appendix Fig S1), as detailed in Appendix Fig S2, and acquired isotope‐labeled synthetic versions of these peptides (AQUA peptides; Gerber et al , 2003).…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Quantitative analysis of human centrosome architecture by targeted proteomics and fluorescence imaging

et al. 2016

View full text Add to dashboard Cite

Centrioles are essential for the formation of centrosomes and cilia. While numerical and/or structural centrosomes aberrations are implicated in cancer, mutations in centriolar and centrosomal proteins are genetically linked to ciliopathies, microcephaly, and dwarfism. The evolutionarily conserved mechanisms underlying centrosome biogenesis are centered on a set of key proteins, including Plk4, Sas‐6, and STIL, whose exact levels are critical to ensure accurate reproduction of centrioles during cell cycle progression. However, neither the intracellular levels of centrosomal proteins nor their stoichiometry within centrosomes is presently known. Here, we have used two complementary approaches, targeted proteomics and EGFP‐tagging of centrosomal proteins at endogenous loci, to measure protein abundance in cultured human cells and purified centrosomes. Our results provide a first assessment of the absolute and relative amounts of major components of the human centrosome. Specifically, they predict that human centriolar cartwheels comprise up to 16 stacked hubs and 1 molecule of STIL for every dimer of Sas‐6. This type of quantitative information will help guide future studies of the molecular basis of centrosome assembly and function.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Quantitative analysis of human centrosome architecture by targeted proteomics and fluorescence imaging

et al. 2016

View full text Add to dashboard Cite

show abstract

“…We have used the selected reaction monitoring (SRM) technique, because it is extensively validated 28,60 , sensitive and, unlike the native mass spectrometry, robust and applicable for inhomogeneous samples. This is particularly advantageous for complexes for which no established purification protocol exists and where the stability of the target complex is unknown (for example, ribosomes from an organism which has not been studied in detail).…”

Section: Methodsmentioning

confidence: 99%

Evolution of the protein stoichiometry in the L12 stalk of bacterial and organellar ribosomes

et al. 2013

View full text Add to dashboard Cite

The emergence of ribosomes and translation factors is central for understanding the origin of life. Recruitment of translation factors to bacterial ribosomes is mediated by the L12 stalk composed of protein L10 and several copies of protein L12, the only multi-copy protein of the ribosome. Here we predict stoichiometries of L12 stalk for 41,200 bacteria, mitochondria and chloroplasts by a computational analysis, and validate the predictions by quantitative mass spectrometry. The majority of bacteria have L12 stalks allowing for binding of four or six copies of L12, largely independent of the taxonomic group or living conditions of the bacteria, whereas some cyanobacteria have eight copies. Mitochondrial and chloroplast ribosomes can accommodate six copies of L12. The last universal common ancestor probably had six molecules of L12 molecules bound to L10. Changes of the stalk composition provide a unique possibility to trace the evolution of protein components of the ribosome.

show abstract

“…3. Specifically, the steps are (1) generation of a hypothesis, a target list of proteins to test the hypothesis and a fit‐for‐purpose quantitation strategy; (2) study design and experimental planning; (3) sample preparation; (4) method refinement; (5) data acquisition; (6) analysis and modeling 34, 35, 36. Bioinformatics and computational proteomics are part of each step of the workflow.…”

Section: Targeted Ms Considerationsmentioning

confidence: 99%

“…All other steps of the SRM‐MS method development have been described in detail elsewhere 36, 39. Figure 3 depicts several of these steps and typical downstream analyses.…”

Section: Targeted Ms Considerationsmentioning

confidence: 99%

Applications of targeted proteomics in systems biology and translational medicine

et al. 2015

Self Cite

View full text Add to dashboard Cite

Biological systems are composed of numerous components of which proteins are of particularly high functional significance. Network models are useful abstractions for studying these components in context. Network representations display molecules as nodes and their interactions as edges. Because they are difficult to directly measure, functional edges are frequently inferred from suitably structured datasets consisting of the accurate and consistent quantification of network nodes under a multitude of perturbed conditions. For the precise quantification of a finite list of proteins across a wide range of samples, targeted proteomics exemplified by selected/multiple reaction monitoring (SRM, MRM) mass spectrometry has proven useful and has been applied to a variety of questions in systems biology and clinical studies. Here, we survey the literature of studies using SRM‐MS in systems biology and clinical proteomics. Systems biology studies frequently examine fundamental questions in network biology, whereas clinical studies frequently focus on biomarker discovery and validation in a variety of diseases including cardiovascular disease and cancer. Targeted proteomics promises to advance our understanding of biological networks and the phenotypic significance of specific network states and to advance biomarkers into clinical use.

show abstract

Selected reaction monitoring–based proteomics: workflows, potential, pitfalls and future directions

Cited by 1,163 publications

References 115 publications

Quantitative analysis of human centrosome architecture by targeted proteomics and fluorescence imaging

Quantitative analysis of human centrosome architecture by targeted proteomics and fluorescence imaging

Evolution of the protein stoichiometry in the L12 stalk of bacterial and organellar ribosomes

Applications of targeted proteomics in systems biology and translational medicine

Contact Info

Product

Resources

About