Peptides stable isotope labeling

Figure 3.2. Stable isotope labeling for quantifying differential protein expression. Cell populations are grown in either 14N or 15N containing medium. Protein lysates are fractionated and separated by 2D gel electrophoresis. Protein spots are excised, digested with trypsin and the mass of the resulting peptides is determined by mass spectrometry. The presence of 15N results in a shift and creates two peaks for each peptide. The ratio of intensities of the peaks is indicative of the relative expression levels of the proteins. Spot A contains a protein that is expressed at similar levels in both cell pools. Spot B contains a protein that is expressed at higher levels in cell pool 2. Figure adapted from Oda et al. (1999).

Figure 16.6 The solid phase ICAT reagent provides a thiol-reactive iodoacetyl group to capture cysteine peptides, a spacer containing stable isotopic labels, and a photo-cleavable group that can release the captured peptides for mass spec analysis. The VICAT mass tag is a solution phase labeling agent that also has a photo-cleavable site to release isolated peptides from a (strept)avidin affinity resin. This compound adds a fluorescent group to better detect labeled peptides as they are being isolated from a sample.

MacCoss, M.J., Wu, C.C., Matthews, D.E. and Yates, J.R., 3rd (2005) Measurement of the isotope enrichment of stable isotope-labeled proteins using high-resolution mass spectra of peptides. Anal. Chem. 77,7646-7653. 

Relative protein quantitation is the basis of all types of differential proteome analyses. In the 2D-gel approach protein staining with either visible or fluorescent dyes provides a reliable and sensitive method to detect changes in protein expression or isoform abundance. In the multidimensional LC approach quantitation relies mostly on stable isotope labeling and ratios between light and heavy isotopomers are determined by MS or MS/MS at the peptide level. Labeling can be performed on the protein level by... 

Procednres have been reported to selectively eiuich samples for specific proteins and/or to fractionate proteomes [16]. This may involve fractionation of cells in subcellular fractions, e.g., organelles, or isolation of protein complexes and/or specific protein emichment strategies. Strategies for reveahng low-abnndance proteins have been reviewed [17]. In addition to prefractionation procednres, apphed prior to LC-MS or 2D-LC-MS, stable-isotope labelling to some extent facilitates selection of specific peptides within the mass spectrometer (Ch. 18.4.1-2). 

As far as protein-related biomarkers are considered, an important tool in biomarker discoveiy is quantitative protein-expression profiling (Ch. 18.4). Mixtures of nonlabelled and stable-isotopically-labelled peptides derived from two different states, e.g., a healthy and disease state, or with and without treatment, are analysed to search for up- or down-regulated protein concentrations. One may question... 

D.R. Goodlett, A. Keller, J.D. Watts, R. Newitt, E.C. Yi, S. Purvine, J.K. Eng, P. von Haller, R. Aebersold, E. Kolker, Differential stable isotope labeling of peptides for quantitation and de novo sequence derivatization. Rapid Commun. Mass Spectrom., 15 (2001) 1214. 

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