High‐resolution high‐performance liquid chromatography with electrospray ionization mass spectrometry and tandem mass spectrometry characterization of a new isoform of human salivary acidic proline‐rich proteins named Roma‐Boston Ser₂₂(Phos) → Phe variant

Abstract

During a survey of human saliva by a top-down reversed-phase high-performance liquid chromatography with electrospray ionization mass spectrometry approach, two proteins eluting at 27.4 and 28.4 min, with average masses of 15 494 ± 1 and 11 142 ± 1 Da, were detected in a subject from Boston. The Δmass value (4352 Da) of the two proteins was similar to the difference in mass values between intact (150 amino acids, [a.a.]) and truncated acidic proline-rich proteins (aPRPs; 106 a.a.) suggesting an a.a. substitution in the first 106 residues resulting in a strong reduction in polarity, since under the same experimental conditions aPRPs eluted at ~22.5 min (intact) and 23.5 min (truncated forms). Manual inspection of the high-resolution high-performance liquid chromatography with electrospray ionization tandem mass spectra of the truncated isoform showed the replacement of the phosphorylated Ser-22 in PRP-3 with a Phe residue. Inspection of the tandem mass spectra of the intact isoform confirmed the substitution, which is allowed by the code transition TCT→TTT and is in agreement with the dramatic increase in elution time. The isoform was also detected in two other subjects, one from Boston (unrelated to the previous) and one from Rome. For this reason we propose to name this variant PRP-1 (PRP-3) RB (Roma-Boston) Ser22(phos)→Phe.

Salivary proline‐rich proteins and gluten: Do structural similarities suggest a role in celiac disease?

“…More detailed analysis with RP‐HPLC‐ESI‐MS of basic PRP isoforms further confirmed the similarity of salivary PRP peptide characteristics in CD/RCD compared to controls. Using anionic PAGE, in two subjects an isoform was noted with lower electrophoretic mobility compared to normal PRP1/3, which we previously reported and named PRB1/3 RB variants (Roma‐Boston Ser22 (Phos) →Phe variant) . The variant was observed in one RCD and one HC, and was thus unlikely to be linked to CD/RCD pathogenesis.…”

“…indicated is in the secreted protein (without signal peptide); information derived from www.uniprot.org and Iavarone et al[61].…”

The Post-Translational Modifications of Human Salivary Peptides and Proteins Evidenced by Top-Down Platforms

“…The di- phosphorylated components (the most abundant) are commonly phosphorylated on Ser 8 and Ser 22 , but a very small percentage of aPRPs phosphorylated on Ser 17 and Ser 22 could be present. The hierarchical phosphorylation on Ser 17 is further substantiated by a study characterizing the aPRP-1 Roma–Boston Ser 22 (phos) → Phe variant, which was never detected as di-phosphorylated proteoform, also using high-resolution HPLC-MS apparatus [ 85 ]. Statherin is mainly di-phosphorylated on Ser 2 and Ser 3 but with the N-terminal sequence being DS 2 S 3 EE…, the two phosphorylation sites are independent and the phosphorylated Ser of the mono-phosphorylated proteoform, always detectable in adult human saliva, can be either Ser 2 or Ser 3 .…”

Saliva, a bodily fluid with recognized and potential diagnostic applications

“…This huge polymorphism of PRPs is further complicated by pre‐secretory cleavages of the pre‐pro‐proteins, (for PRB1, PRB2, PRB4 loci) generating smaller peptides, by the existence of SNPs coding for variants (for PRH2 and PRB1‐4 loci) and splicing variants (for PRB1 locus). Detailed descriptions of all these possibilities are reported in several recent references [ 75 , 76 , 77 ].…”

“…Unfortunately, different biomarkers are sometimes suggested for the same pathology even when similar platforms are applied, and this generates legitimate doubts about the strength of the experimental plan, the adequacy of the number of samples under study, and the choice of controls [ 78 ]. Moreover, in proteomic platforms, the increased number of components under observation, strongly enhances the detection of variations connected to an interindividual polymorphism and not to a disease and, as above reported, human saliva is a bodily fluid displaying relevant polymorphisms [ 73 , 74 , 75 , 76 , 77 ]. As an example, proteomic analysis of saliva has been extensively utilized to reveal biomarkers of Sjögren's syndrome, suggesting a panel of proteins all linked to the inflammatory phase and these results have been largely reviewed [ 79 , 80 ].…”