In Silico Peptide Repertoire of Human Olfactory Receptor Proteomes on High-Stringency Mass Spectrometry

Human olfactory receptors
(ORs) are seven-pass transmembrane G-protein
coupled receptors (GPCR) involved in smell perception and many other
signaling pathways. They are primarily expressed in the olfactory
epithelium and ectopically expressed in several other organs and tissues.
neXtProt contains 4 PE1 (protein existence 1, evidenced at the protein
level) ORs, determined on the basis of either protein interaction
data (i.e., OR1D4 and OR2AG1) or convincing genetic, haplotype, or
biochemical data (i.e., OR1D2 and OR2J3). Not a single OR currently
qualifies for neXtProt PE1 status based on mass spectrometry (MS)
evidence. Many reasons for this absence of MS-based identification
have been proposed, including (i) confined or spatiotemporal or developmental
expression, (ii) low copy number, (iii) OR repertoire gene silencing,
and (iv) limited tissue availability. OR transmembrane domains (TMDs)
inherently limit MS identification because the hydrophobic nature
restricts the access of trypsin to potential cleavage sites. Equally,
the extremely low frequency or lack of accessible arginine and lysine
residues in TMDs renders trypsin cleavage ineffective. Here, we demonstrate
an analytical approach specifically focused on the hydrophilic (trypsin-accessible)
domains of ORs [i.e., with all transmembrane segments and anchored
peptides excluded). We predicted the ability of OR soluble (hydrophilic)
domains to yield 2 or more >9 amino acids (aa) length unique mapping
(unique to a protein only), non-nested (peptides with varying length
at the N or C terminal but containing the same core sequence), leucine/isoleucine
(I/L) switch examined (I and L have same mass and cannot be distinguished
by MS) tryptic peptides. Our analysis showed that ∼58% of the
human OR proteome could potentially generate tryptic peptides that
satisfy current the Human Proteome Project data interpretation guidelines
(version 2.1) when no missed cleavages are allowed and increases to
∼78% when one missed cleavage is allowed. The utilization of
current biological data (adjuvant genomics, expression profile, transcriptomics,
epigenome silencing data, etc.) and the adoption of a non-conventional
proteomics approach (e.g., Confetti multiprotease digestion, CNBr
cleavage of TMDs, and more-extreme chromatographic and MS methods)
could aid in the detection of the remaining ORs.