Abstract
N-terminal processing of proteins is a process affecting a large part of the eukaryotic proteome. Although N-terminal processing is an essential process, not many large inventories are available, in particular not for human proteins. Here we show that by using dedicated mass spectrometry-based proteomics techniques it is possible to unravel N-terminal processing in a semicomprehensive way. Our multiprotease approach led to the identification of 1391 acetylated human protein N termini in HEK293 cells and revealed that the role of the penultimate position on the cleavage efficiency by the methionine aminopeptidases is essentially conserved from Escherichia coli to human. Sequence analysis and comparisons of amino acid frequencies in the data sets of experimentally derived N-acetylated peptides from Drosophila melanogaster, Saccharomyces cerevisiae, and Halobacterium salinarum showed an exceptionally higher frequency of alanine residues at the penultimate position of human proteins, whereas the penultimate position in S. cerevisiae and H. salinarum is predominantly a serine. Genome-wide comparisons revealed that this effect is not related to protein N-terminal processing but can be traced back to characteristics of the genome.
Highlights
N-terminal processing of proteins is a process affecting a large part of the eukaryotic proteome
Most N-acetylations are accomplished by N-terminal acetyltransferase (NAT)1 complexes of which some are known to associate with the ribosome complexes [6]
Low pH SCX has been proven to enrich for N-acetylated peptides from a pool of “regular” tryptic peptides, exploiting the fact that N-acetylated peptides have one less positive charge in solution due to the blocked N terminus (13, 26 –28)
Summary
N-terminal processing of proteins is a process affecting a large part of the eukaryotic proteome. This modification can occur on the ultimate methionine residue, which forms the main target of acetylation, or after the cleavage of the N-terminal methionine residue Together, these modifications occur on the vast majority of eukaryotic proteins [1, 2]. Most of the current insights into sequence specificity for N-acetylation comes from studies using yeast strains in which specific NAT genes were deleted In these studies the substrate specificities for the yeast acetyltransferases (Ard1p, Nat3p, and Mak3p) were deduced from the lack of acetylation of protein subsets in the different yeast knock-out strains.
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