Abstract
Acetylation of nascent protein Nα-termini is a common modification among archae and eukaryotes and can influence the structure and function of target proteins. This modification has been studied on an individual protein or (synthetic) peptide level or on a proteome scale using two-dimensional polyacrylamide gel electrophoresis. We recently developed mass spectrometry driven proteome analytical approaches specifically targeting the amino (N) terminus of proteins based on the concept of diagonal reverse-phase chromatography. We here review how this so-called combined fractional diagonal chromatography (COFRADIC) technique can be used in combination with differential mass-tagging strategies as to both qualitatively and quantitatively assess protein Nα-acetylation in whole proteomes.
Highlights
Earlier proteome analyses aiming at characterizing the protein N-acetylation status primarily used two-dimensional protein gels to compare maps of acetylated and non-acetylated proteins
Data generated from such studies added a lot to our understanding of the biology of protein Nα-terminal acetylation it was realized that intrinsic drawbacks of 2D polyacrylamide gel electrophoresis techniques could result in sketchy proteome maps in which mainly well-soluble and highly abundant proteins were detected
Full technical details on the isolation of N-terminal peptides using the COFRADIC technology are documented elsewhere [11] and we here only describe additional chemistries that lead to quantification of the degree of protein acetylation, segregation of the in vivo blocked and free variants of protein N-terminal peptides and enrichment of methionine-containing N-terminal peptides
Summary
Earlier proteome analyses aiming at characterizing the protein N-acetylation status primarily used two-dimensional protein gels to compare maps of acetylated and non-acetylated proteins. Studies comparing the proteomes of wild-type yeast with mutant strains deficient for a specific N-acetyltransferase (NAT) complex revealed charge shifts due to the absence or the presence of positively charged α-amino groups [1]. Data generated from such studies added a lot to our understanding of the biology of protein Nα-terminal acetylation it was realized that intrinsic drawbacks of 2D polyacrylamide gel electrophoresis techniques could result in sketchy proteome maps in which mainly well-soluble and highly abundant proteins were detected. Some gel-free techniques enrich protein N-terminal peptides independent of their in vivo modification status (blocked or free) and (page number not for citation purposes)
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