pSNAP: Proteome-wide analysis of elongating nascent polypeptide chains

Abstract

Cellular global translation is often measured using ribosome profiling or quantitative mass spectrometry, but these methods do not provide direct information at the level of elongating nascent polypeptide chains (NPCs) and associated co-translational events. Here we describe pSNAP, a method for proteome-wide profiling of NPCs by affinity enrichment of puromycin- and stable isotope-labeled polypeptides. pSNAP does not require ribosome purification and/or chemical reaction, and captures bona fide NPCs that characteristically exhibit protein N-terminus-biased positions. We applied pSNAP to evaluate the effect of silmitasertib, a potential molecular therapy for cancer and COVID-19 patients, and revealed acute translational repression through casein kinase II and mTOR pathways. We also characterized modifications on NPCs and demonstrated that the combination of different types of modifications, such as acetylation and phosphorylation in the N-terminal region of histone H1.5, can modulate interactions with ribosome-associated factors. Thus, pSNAP provides a framework for dissecting co-translational regulations on a proteome-wide scale.

Monitoring mitochondrial translation by pulse SILAC

“…Quantitative non-canonical amino acid tagging (QuaNCAT) (Eichelbaum et al, 2012; Howden et al, 2013; Schanzenbächer et al, 2016) relies on pulse labeling of newly synthesized proteins with a methionine analog, azidohomoalanine (AHA) (Dieterich et al, 2006), allowing for selective enrichment of the tagged protein pool through click-chemistry as well as MS-based profiling of the tagged proteins. Nascent chain proteomics using puromycin or its analogs enables isolation and identification of nascent polypeptide chains that are being elongated by the ribosomes (Aviner et al, 2013; Forester et al, 2018; Hünten et al, 2015; Schäfer et al, 2022; Tong et al, 2020; Uchiyama et al, 2020, 2021). However, these methods require a large number of cells (typically >10 7 cells), involve multiple steps to purify newly synthesized proteins via affinity purification and/or require the isolation of ribosome complexes through density gradient ultracentrifugation.…”

Monitoring mitochondrial translation by pulse SILAC

“…Quantitative noncanonical amino acid tagging ( 14 , 15 , 16 ) relies on pulse labeling of newly synthesized proteins with a methionine analog, azidohomoalanine (AHA) ( 17 ), allowing for selective enrichment of the tagged protein pool through click chemistry as well as MS-based profiling of the tagged proteins. Nascent chain proteomics using puromycin or its analogs enables isolation and identification of nascent polypeptide chains that are being elongated by the ribosomes ( 18 , 19 , 20 , 21 , 22 , 23 , 24 ). However, these methods require a large number of cells (typically >10 7 cells), involve multiple steps to purify newly synthesized proteins via affinity purification, and/or require the isolation of ribosome complexes through density gradient ultracentrifugation.…”