Annotating N Termini for the Human Proteome Project: N Termini and Nα-Acetylation Status Differentiate Stable Cleaved Protein Species from Degradation Remnants in the Human Erythrocyte Proteome

A goal
of the Chromosome-centric Human Proteome Project is to identify
all human protein species. With 3844 proteins annotated as “missing”,
this is challenging. Moreover, proteolytic processing generates new
protein species with characteristic neo-N termini that are frequently
accompanied by altered half-lives, function, interactions, and location.
Enucleated and largely void of internal membranes and organelles,
erythrocytes are simple yet proteomically challenging cells due to
the high hemoglobin content and wide dynamic range of protein concentrations
that impedes protein identification. Using the N-terminomics procedure
TAILS, we identified 1369 human erythrocyte natural and neo-N-termini
and 1234 proteins. Multiple semitryptic N-terminal peptides exhibited
improved mass spectrometric identification properties versus the intact
tryptic peptide enabling identification of 281 novel erythrocyte proteins
and six missing proteins identified for the first time in the human
proteome. With an improved bioinformatics workflow, we developed a
new classification system and the Terminus Cluster Score. Thereby
we described a new stabilizing N-end rule for processed protein termini,
which discriminates novel protein species from degradation remnants,
and identified protein domain hot spots susceptible to cleavage. Strikingly,
68% of the N-termini were within genome-encoded protein sequences,
revealing alternative translation initiation sites, pervasive endoproteolytic
processing, and stabilization of protein fragments in vivo. The mass
spectrometry proteomics data have been deposited to ProteomeXchange
with the data set identifier .