The Human Dental Pulp Proteome and N‑Terminome: Levering the Unexplored Potential of Semitryptic Peptides Enriched by TAILS to Identify Missing Proteins in the Human Proteome Project in Underexplored Tissues

An underexplored yet widespread feature
of the human proteome is
the proteolytic proteoforms of proteins. We used terminal amine isotopic
labeling of substrates (TAILS), a high-content N-terminal positional
proteomics technique, for in-depth characterization of the human dental
pulp proteome from its N-terminome and to provide data for the Chromosome-centric
Human Proteome Project (C-HPP). Dental pulp is a unique connective
tissue maintaining tooth sensation and structure by supporting a single
cell layer of odontoblasts that synthesize mineralization-competent
dentine extracellular matrix. Therefore, we posited pulp to be a rich
source of unique tissue-specific proteins and hence an abundant source
of “missing” proteins as defined by neXtProt. From the
identified 4332 proteins (false discovery rate (FDR) ≤ 0.7%),
21 528 unique peptides (FDR ≤ 1.0%) and 9079 unique
N-termini, we analyzed N-terminal methionine excision, co- and posttranslational
Nα-acetylation, protein maturation, and proteolytic processing.
Apart from 227 candidate alternative translation initiation sites,
most identified N-termini (78%) represented proteolytic processing
and mechanism-informative internal neo-N-termini, confirming a pervasive
amount of proteolytic-processing generated proteoforms in vivo. Furthermore,
we identified 17 missing protein candidates for the C-HPP, highlighting
the importance of using (i) less studied human specimens and (ii)
orthogonal proteomic approaches such as TAILS to map the human proteome.
The mass spectrometry raw data and metadata have been deposited to
ProteomeXchange with the PXD identifier .