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 <PXD002264>.