sample + new; affinity purification; sample + new and affinity purification; nacent protein

Figure 2. Mass Spectrometry-Based Methods for Measuring Differential Translation Changes Across Proteome. (A) Pooling proteins from two samples, in the pulsed stable isotope labeling by amino acid in cell culture (pSILAC) method, provides quantitative mass spectrometry of protein produced during the labeling period. (B) In bio-orthogonal noncanonical amino acid tagging (BONCAT), CLICK-reactive bio-orthogonal amino acids [e.g., azidohomoalanine (AHA)] are incorporated into the protein and subsequent biotin tagging allows affifinity purifification of newly synthesized protein for wider coverage of the proteome. (C) Quantitative noncanonical amino acid tagging (QuaNCAT) combines pSILAC and BONCAT for quantitative mass spectrometry with broader coverage. (D) Puromycin-associated nascent chain proteomics (PUNCH-P) captures polypeptides from active translation through biotin-conjugated puromycin and subsequent purifification by streptavidin beads. Metabolic pulse labeling of newly synthesized proteins with stable isotopes offers a solution to this problem, enabling quantitative analysis of protein synthesis and decay. In pulsed stable isotope labeling by amino acid in cell culture (pSILAC) [5,18], arginine and lysine comprising heavy nitrogen, carbon, or hydrogen isotopes, are placed into tissue culture media to mark the protein translated during the given time. Two different compositions of labeled amino acids enable the labeling of proteins with ‘heavy’ and ‘medium-heavy’ molecular weight. Pooling those two differentially labeled samples together and performing mass spectrometry on the mixture yields a quantitative comparison of the translation status in two different conditions (i.e., ‘heavy’ versus ‘medium-heavy’), distinguishing new proteins in each sample from pre-existing ‘light’ proteins (Figure 2A). To achieve broader proteome coverage, the isolation and enrichment of newly synthesized protein are advantageous for enabling deeper mass spectrometry analysis, which can be limited by interference from more abundant pre-existing proteins and other detectability biases. Here again, CLICK chemistry offers a solution: after bio-orthogonal noncanonical amino acid tagging (BONCAT), a CLICK reaction with alkyne-conjugated biotin allows purifification with streptavidin/neutravidin-beads [19,20] (Figure 2B). BONCAT can be combined with pSILAC, in an approach termed ‘quantitative noncanonical amino acid tagging’ (QuaNCAT), which allows quantitative, ratiometric, and deep proteomic analysis of newly synthesized protein [21] (Figure 2C). Moreover, cell-selective BONCAT/QuanCAT can be performed using cell type- specifific expression of an engineered aminoacyl-tRNA synthetase (tRS) for charging azide- bearing bio-orthogonal amino acids, which are a poor substrate for nonengineered tRS in their original form [22–26]. Alternatively, in vitro puromycylation with a biotin-conjugated puromycin derivative can label nascent proteins in the cell extract, which can then be enriched by streptavidin affifinity purifification. Proteomic analysis of these tagged translation intermediates is termed ‘puromycin-associated nascent chain proteomics’ (PUNCH-P) [27] (Figure 2D).