Top-down platform for deciphering the human salivary proteome

Abstract

Proteomic platforms can be classified in bottom-up strategies, which analyze the sample after proteolytic digestion, and top-down strategies, which analyze the intact naturally occurring proteome. Bottom-up platforms are high-throughput because they can investigate a large number of proteins, regardless of their dimension. Nonetheless, information on post-translational modifications (PTMs) can be lost, especially those regarding naturally occurring cleavages and alternative splicing. Top-down platforms cannot cover vast proteomes, however, they can disclose subtle structural variations occurring during protein maturation and allow label-free relative quantifications in an unlimited number of samples. A repertoire of 256 masses belonging to naturally occurring proteins and peptides consistently detected by RP-HPLC-ESI-MS analysis of the acidic soluble fraction of human whole saliva is presented in this study. Of them, 233 have been identified, while 23 are still pending for the definitive characterization. The present review reports average and mono-isotopic masses of the peptides and proteins detected, RP-HPLC elution times, PTMs, origin and quali-quantitative variations observed in several physiological and pathological conditions. The information reported can be a reference for users of top-down RP-HPLC-ESI-MS proteomic platforms applied to the study of the human salivary proteome as well as of other human bodily fluids.

Chrono-Proteomics of Human Saliva: Variations of the Salivary Proteome during Human Development

“…20,21 From our data we assumed that HIS1 protein expression started around 7 months (210 days) ± 2 weeks of PCA. Furthermore, as already reported in a recent 13,19 The nomenclature of the different bPRPs was described by Castagnola et al 13 study, we confirmed that Hst1 showed two maxima of concentrations around 1 and 6 years of age, in correspondence with deciduous and permanent dentitions. 11 Concerning Hst3 and its derivatives histatin 6 (Hst6) and histatin 5 (Hst5), the present study suggested that HIS2 protein expression commonly starts later than HIS1 roughly after the normal term of delivery (270−280 days of PCA).…”

“…The majority of individuals in the Western population express five major isoforms of aPRPs: PIF-s, Db-s (where "s" stands for slow), and Pa (commonly detected as 2-mer) encoded by the PRH1 locus and PRP-1 and PRP-2 encoded by the PRH2 locus. 18 13,19 As evidenced in Figure 2, bPRPs encoded by PRB1, PRB2, and PRB4 genes are only detectable as multiple peptide fragments generated by presecretory cleavages of the pro-proteins, and the connection between the most common haplotypes and salivary phenotypes is still pending complete definition. 13,14,19 Expression of some bPRPs occurs later with respect to all of the other salivary proteins.…”

“…IB-1 and II-2 are two bPRP peptides, 96 and 75 residues long, respectively, closely eluting in the HPLC−ESI−MS profile and sharing very similar sequences and both phosphorylated on Ser 8 . 13,19 These bPRPs are undetectable in whole saliva before 4−6 years of age. When revealed, the phosphorylated proteoforms were the prevalent ones, being the nonphosphorylated sporadically detected, as in the adults (data not reported).…”

Anti-tumoral activity of human salivary peptides

“…2. According to available data [8], the isolated peptides correspond to salivary proline-rich peptide IB-4 (Uniprot accession number P02812) and histidine-rich peptide 1 (Uniprot accession number P15515), both belonging to groups of peptides described as having potential anti-tumoral/anti-microbial activity [10]. Importantly, the mass fingerprints showed that the selected fractions exhibited a high degree of purity and, consequently, any cytotoxic effects will most probably be due to the peptides present in these fractions.…”

“…Table 1 highlights the sequence of the peptides used in the present study. These were selected based on previous studies underlining their abundance in human saliva [8,15].…”