Identification of N-degrons and N-recognins using peptide pull-downs combined with quantitative mass spectrometry

Methods Enzymol. 2023:686:67-97. doi: 10.1016/bs.mie.2023.02.007. Epub 2023 Mar 24.

Abstract

Regulated protein degradation controls protein levels of all short-lived proteins to ensure cellular homeostasis and also protects cells from misfolded or other abnormal proteins. The most important players in the degradation system are E3 ubiquitin ligases which recognize exposed sequence motifs, so-called degrons, of target proteins and mark them through the attachment of ubiquitin for degradation. N-terminal (Nt) sequences are extensively used as degrons (N-degrons) and all 20 amino acids are able to feed proteins in 1 of the 5 known N-degron pathways. Studies have mainly focused on characterizing systematically the role of the starting amino acid on protein stability and less on the identification of the E3 ligases involved. Recent data from our lab and literature suggest that there is an extensive interplay of N-recognins and Nt-modifying enzymes like Nt-acetyltransferases (NATs) or N-myristoyltransferases which only starts to be elucidated. It suggests that improperly modified or unexpectedly unmodified proteins become rapidly removed after synthesis ensuring protein maturation and quality control of specific subsets of proteins. Here, we describe a peptide pull-down and down-stream bioinformatics workflow conducted in the MaxQuant and Perseus computational environment to identify N-recognin candidates in an unbiased way using quantitative mass spectrometry (MS)-based proteomics. Our workflow allows the identification of N-recognin candidates for specific N-degrons, to determine their sequence specificity and it can be applied as well more general to identify binding partners of N-terminal modifications. This method paves the way to identify pathways involved in protein quality control and stability acting at the N-terminus.

Keywords: E3 ligase; Label-free quantification; Mass-spectrometry; N-degron; N-recognin; N-terminal acetylation; N-terminal modification; Peptide pull-down; Protein quality control and stability; Quantitative MS-based proteomics.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Mass Spectrometry
  • Peptides* / chemistry
  • Proteolysis
  • Ubiquitin / metabolism
  • Ubiquitin-Protein Ligases* / metabolism

Substances

  • recognins
  • Peptides
  • Ubiquitin-Protein Ligases
  • Ubiquitin