Quantification Strategies

In general, quantification can be achieved by two main strategies - use of stable isotope labeling or without, so-called “label-free" The principle of labeling-based quantitative proteomics is the introduction of heavy stable isotopes such as 2H, 13C, and 15N to proteins, which can be discriminated from unlabeled proteins for comparative analysis. The introduction of an isotope label results in a predictable mass difference between a labeled peptide and the unlabeled counterpart, and thus stable isotope labeling provides a means for sample comparison in the same analytical LC-MS run. Stable isotope quantification can be achieved via metabolic or chemical labeling. Stable isotope labeling of amino acids in cell culture (SILAC and related variants) is a widely used form of metabolic labeling, typically employing heavy lysine and arginine for protein labeling. SILAC achieves MS1 level quantification: peptides to be compared exist in heavy (labeled) and light (unlabeled) forms, for the simplest implementation in duplex format. As previously discussed (Section 4.2.2), heavy methyl SILAC and isomethionine (i) methyl SILAC can be used for analysis of protein methylation [39, 46]. In contrast, chemical labeling with isobaric tags provide MS2 level relative quantification. Examples include the isobaric tag for relative and absolute quantification (iTRAQ) and tandem mass tag (TMT) reagents. iTRAQ (4 plex, 8 plex) and TMT (2, 6, and 10 plex) labeled peptide sets coelute to form a single MS1 peak, and quantification of the component peptides is achieved by measurement of distinct reporter ions released upon peptide fragmentation. SILAC, iTRAQ, TMT, and label-free methods are typically used to quantify proteome and protein subsets [52]. Specific enrichment strategies increase the sensitivity of analysis of methylated and acetylated peptide subsets of the proteome (see Section 4.4). Isotope labeling can be applied to determine PTM stoichiometry as described later (see Section

Quantitative MS can also be performed in a label-free manner by comparing peptide intensities in a series of m/z and retention time-aligned LC-MS runs. The approach requires high interrun reproducibility, since each sample is run individually. Label-free methods are based on MS2 or MS1 level intensity information. For MSl-based quantification methods, the area under the curve from the extracted ion chromatogram of the peptide precursor provides a quantitative measure, with MS2 data used for the identification of peptides. MS1 quantification assumes that each precursor is a single species and cannot discriminate isobaric peptides; the availability of MS2 data is very useful where unique products provide a means to discriminate. Spectral counting and intensity-based measurements of peptide fragments at the MS2 level provide a measure of protein abundance, but this MS2 level quantification is not generally applied to PTM peptides [53]. Label-free methods enable sample comparison based on relative or absolute quantification. Label-free methods can be used for discovery and targeted proteomics, while absolute quantification is widely applied to targeted proteomics.

Absolute quantification can be achieved by the inclusion of a synthetic reference (AQUA) peptide, that is, same sequence as peptide of interest with heavy label, for example, 13C, spiked at a known concentration into the sample to be analyzed. The synthetic AQUA peptide and corresponding peptide of interest have the same physicochemical properties and thus coelute and share ionization and fragmentation characteristics but can be distinguished on the basis of (known) mass difference [54].

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