b'Protein quantitation using massSample preparationspectrometrySample preparation is one of the most variable and time-Differences in protein expression can be studied both globallyconsuming steps in the analysis of proteins by MS, and the (discovery proteomics) or within a specific subset of proteinsquality and reproducibility of sample extraction and preparation (targeted proteomics). Most quantitative proteomic analysessignificantly impact the results. Robust, integrated workflows utilize the isotopic labeling of proteins or peptides in theenable consistent results between labs and help eliminate wasted experimental groups, which can then be differentiated bytime spent troubleshooting experimental methods and results.mass spectrometry. Relative quantitation methods are usedProtein extraction, depletion, and enrichment strategies have to compare protein or peptide abundance between samples,been developed to remove high-abundance proteins or isolate while spiking unlabeled samples with known concentrationstarget proteins in the sample, reduce sample complexity, and of isotopically labeled synthetic peptides can help to enablehelp improve the detection of low-abundance proteins. Digestion absolute quantitation of target peptides via selected reactionis required because proteins are often too big and complex for monitoring (SRM). analysis and therefore are digested into peptides for MS analysis Discovery quantitation strategies for relative protein quantitationfor better detection and identification of proteins. Trypsin is the include stable isotope labeling using amino acids in cell cultureprotease of choice for protein digestion. However, digestion (SILAC) and labeling using tandem mass tag (TMT) reagents.with alternative proteases such as Glu-C, Lys-N, Lys-C, Asp-N, SILAC-based quantitation involves metabolically labeling proteinor chymotrypsin can improve individual protein sequence samples in cultured mammalian cells with a heavy isotope coverage or generate unique peptide sequences for different labeled form of an amino acid. Inclusion of the labeled aminoMS applications. acid in cell or tissue culture media results in replacement of the natural light amino acid with the heavy form in newly synthesizedInstrument calibrationproteins, enabling multiplexing. Isobaric chemical tags are a moreCalibration solutions and standards are critical for optimal universal alternative to SILAC because they can be used with aperformance in mass spectrometry; mass spectrometers must wide variety of samples including cells, tissues, and biologicalbe carefully monitored to ensure accuracy of results. Ideally, fluids. Isobaric chemical tags facilitate the simultaneous analysiscalibration reagents should be ready-to-use liquid formulations of up to eighteen samples and consist of an MS/MS reportercomposed of highly purified, ionizable molecules or polymers group, a spacer arm, and a reactive group. Amine-reactivespecifically designed for positive or negative calibration of groups covalently bind to peptide N termini or to lysine residues.instruments. Standards should be designed for specific Each tag fragments during MS/MS, producing unique reporterapplications, including sensitivity assessment, determination of ions, enabling multiplexed quantitation through comparing thedigestion efficiency, chromatography assessment, or as controls intensities of the reporter ions.for sample analysis.Absolute quantitation is performed in targeted proteomic experiments and increases the sensitivity of detection for a limited number of target analytes. These approaches require spiking a sample with known amounts of synthetic peptides containing heavy stable isotopes, which act as internal quantitative standards for absolute quantitation of the corresponding natural peptides in the sample.88 Mass spectrometry thermofisher.com/proteinbiology'