Systems Analysis of Chromatin-Related Protein Complexes in Cancer by Andrew Emili Jack Greenblatt & Shoshana Wodak

Author:Andrew Emili, Jack Greenblatt & Shoshana Wodak
Language: eng
Format: epub
Publisher: Springer New York, New York, NY

Metabolic Labeling for Characterizing Dynamic Changes in Chromatin Proteins

Label-free techniques based on protein spectral counts often have a limited dynamic range that is offset by metabolic/isotope labeling techniques that can provide more accurate quantification at low signal-to-noise and reduce errors introduced during sample preparation prior to mass spectrometry analysis. A metabolic labeling method, Stable Isotope Labeling by Amino acids in Cell culture (SILAC), which allows the incorporation of stable isotope amino acid residues into proteins has been greatly eased quantitative proteomic analysis. Matched cultures of cells are grown in identical media except that one medium contains a “light” and the other a “heavy” form of a selected amino acid (e.g., 12C- and 13C-labeled L-leucine, respectively). Metabolic incorporation of stable isotope amino acids results in pairs of chemically identical peptides that can be detected by MS. The relative abundance of the two proteins can be accurately determined by the ratio of MS peak intensities for such peptide pairs (Fig. 4). Because of its high sequence coverage of identified proteins, high labeling efficiency, and simplicity of incorporation, this strategy has become one of the most popular methods for quantitative characterization of differentially expressed proteins and posttranslational modifications.

Fig. 4SILAC labeling enables comparative analysis of multiple samples. LC-MS of unlabeled myotubes (blue) and 13C6,15N4-labeled myoblasts (red) shows differential expression of skeletal muscle marker, desmin (TFGGAPGFPLGSPLSSPVFPR) (2+)

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