NAPT, an unbiased approach for sequential analysis of protein N- and C- terminome

ABSTRACT: Robust analysis of both protein N- and C-termini can reveal novel N- and C-degrons that modify protein stability and shape the eukaryotic proteome. Herein, we designed a workflow, termed LysargiNase-assisted Analysis of Protein N- and C-Terminome (NAPT) by sequential and selective separating N- and C-termini on strong cation exchange chromatography (SCX). Taking advantage of N-terminal peptides’ reduced charge states at pH 2.7 and C-terminal peptides’ enhanced charge states at pH 8.5, an adequate shift of pH during SCX fractionation could easily lead to sequential elution of N-terminal, internal, and C-terminal peptides. Combining NAPT with multiple enzymatic digestion strategies, we identified 2458 human protein N-termini and 3056 human protein C-termini from Hela cell line. As N-terminal peptides generally bear two less positive charges than internal peptides at pH 2.7 after LysargiNase digestion, leaving one-positive-charge tolerance for histidine-containing N-terminal peptides, and C-terminal peptides were separated at pH 8.5 where histidine was neutrally charged, NAPT workflow showed minimal bias against histidine residues and excellent complementarity compared to its sister, the SAPT workflow, which adopted trypsin as protease. Taken together, 4285 protein N-termini and 4170 protein C-termini were identified by NAPT and SAPT, representing the largest human protein C-termini and the second largest human protein N-termini dataset so far, demonstrating their valuable potential in terminomic studies. Furthermore, we systematically analyzed sequences of identified protein termini and validated that their behavior obeyed several degron pathways.

ORGANISM(S): Homo Sapiens

SUBMITTER: Haojie Lu  

PROVIDER: PXD030791 | iProX | Mon Apr 11 00:00:00 BST 2022

REPOSITORIES: iProX