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Proteome-wide analysis of elongating nascent polypeptide chains – peptide pulldown

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. We combine quantitative proteomics, pulse labeling with puromycin, and stable isotope-labeled amino acids to analyze thousands of NPCs. Our approach enables global analyses of translational responses and co-translational modifications, providing a framework for dissecting co-translational regulations proteome-wide. The inputs (without IP) were analyzed in triplicates.

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Yasushi Ishihama

PROVIDER: PXD026349 | JPOST Repository | Sat May 28 00:00:00 BST 2022

REPOSITORIES: jPOST

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			Action	DRS
	210325_KI_HS_162_4_SETA_rep1.raw	Raw
	210325_KI_HS_162_4_SETA_rep2.raw	Raw
	210325_KI_HS_162_4_SETA_rep3.raw	Raw
	210325_KI_HS_162_5_Ac-SETA_rep1.raw	Raw
	210325_KI_HS_162_5_Ac-SETA_rep2.raw	Raw

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Publications

pSNAP: Proteome-wide analysis of elongating nascent polypeptide chains.

Uchiyama Junki J Roy Rohini R Wang Dan Ohtan DO Morikawa Kazuya K Kawahara Yuka Y Iwasaki Mio M Yoshino Chiaki C Mishima Yuichiro Y Ishihama Yasushi Y Imami Koshi K

iScience 20220603 7

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 labeling, and captures <i>bona f ...[more]

PMID: 35754732

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Project description: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. We combine quantitative proteomics, pulse labeling with puromycin, and stable isotope-labeled amino acids to analyze thousands of NPCs. Our approach enables global analyses of translational responses and co-translational modifications, providing a framework for dissecting co-translational regulations proteome-wide. HeLa cells were pre-incubated with either DMSO or a CK2 inhibitor CX4945 20 for 10 min, and subsequently, cells were pulse-labeled with puromycin and SILAC amino acids for 2 hr in the presence of DMSO or CX4945, and immunoprecipitation of puromycylated proteins was performed in triplicates.

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Proteome-wide analysis of elongating nascent polypeptide chains – Nt-acetylation analysis

Project description: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. We combine quantitative proteomics, pulse labeling with puromycin, and stable isotope-labeled amino acids to analyze thousands of NPCs. Our approach enables global analyses of translational responses and co-translational modifications, providing a framework for dissecting co-translational regulations proteome-wide. HeLa cells were treated with either CHX or DMSO for 2 hr in the presence of puromycin and corresponding SILAC amino acid pairs. By combining pSNAP with low pH strong cation exchange (SCX) chromatography, we enriched Nt-acetylated peptides that were eluted earlier in SCX fractionation due to the loss of positive charge at their N-terminal ends. Immunoprecipitation of puromycylated proteins was performed in triplicates.

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pSNAP: Proteome-wide analysis of elongating nascent polypeptide chains.

Project description: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 labeling, 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 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.

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