Finding new posttranslational modifications in salivary proline‐rich proteins

Abstract

Proline-rich proteins (PRPs) are the most complex family of salivary peptides with distinct isoforms and PTMs. Up to date, only the serine phosphorylation at positions 8, 17, and 22 have been experimentally observed on acidic PRP (aPRPs), and at position 8 on basic PRP1 and 2. The presence of a glucoronyl group at Ser17 was also noticed on aPRP. The main goal of this study was to identify new PTMs and distinct isoforms of salivary PRPs using LC-MALDI-TOF/TOF. Through the salivary peptidome characterization of 20 different subjects from Control, Diabetic, and Head and Neck Cancer groups, it was possible to identify the following species: (i) N-glycosylation sites: two in basic proline-rich protein 2 (bPRP2), one in bPRP3 and one in bPRP4; (ii) O-glycosylation sites: two in bPRP2 and one in aPRP; (iii) other terminal monosaccharide sites: six in bPRP1, two in bPRP2 and two in bPRP3; (iv) other modifications such as N-terminal pyro-Glu (two in bPRP1, six in bPRP2, eight in bPRP3 and nine in bPRP4); (v) phosphorylation in serine, three in bPRP1, one in bPRP2, one in bPRP3 and one in aPRP1; (vi) bPRP1 (allele S, allele M and variant CP5) and bPRP4 (allele M). In summary, salivary peptidome data analysis allowed the identification of 45 new PRP-modified residues, mainly due to glycosylation, phosphorylation and conversion of Gln to pyro-Glu. Moreover, comparing all subject groups, it was noticed a predominance of N-acetyl hexosamine modification on bPRPs in the Head and Neck Cancer patients.

Integrated Omics and Computational Glycobiology Reveal Structural Basis for Influenza A Virus Glycan Microheterogeneity and Host Interactions

“…We evaluated the NetNGlyc server (89) (http://www.cbs.dtu.dk/services/NetNGlyc/), used in published studies (90–93, 27) to provide a qualitative measure of the likelihood for N -linked glycosylation, given the protein sequence; however, we found that NetNGlyc predictions inconsistent with our results. Table I compares NetNGlyc predictions and site-occupancy results from proteomics data for Phil-82.…”

Automated Mass Spectrometry–Based Functional Assay for the Routine Analysis of the Secretome

“…For example, secreted proteins are glycosylated through both N-and O-glycosidic bonds, and if the peptide backbone is occupied by oligosaccharides, then most likely they will not be identified in a proteomic analysis. 40,[45][46][47][70][71][72] As such, if the glycosylated protein has a low molecular mass, then the chance of being identified decreases. Therefore, the immediate consequence is false-negative data.…”

“…Although this method is much better, more sensitive, and perhaps more reliable providing sequence information, there are also drawbacks associated with this method: Some proteins truncate and/or degrade, and when analyzed, this information is not taken into consideration. [45][46][47]49 As such, if a quantitation is performed, there is a high chance that the quantitation will produce false-positive or not very accurate results. [50][51][52][53][54] To compensate for that, one may have to consider the use of more than two or even three peptides for quantitation purposes.…”

“…Although the principle is simple, this method is not very successful, simply because many eukaryotic cells secrete proteins that are intensely posttranslationally modified. [40][41][42][43][44][45][46][47] Therefore, a peak in the MALDI-MS spectrum does not represent a protein or a peptide but rather a posttranslationally modified protein (or peptide). [45][46][47] However, even if the differences detected are not at the "naked" protein level, the differences observed may still be detected and the proteins responsible for them can be identified.…”

“…[40][41][42][43][44][45][46][47] Therefore, a peak in the MALDI-MS spectrum does not represent a protein or a peptide but rather a posttranslationally modified protein (or peptide). [45][46][47] However, even if the differences detected are not at the "naked" protein level, the differences observed may still be detected and the proteins responsible for them can be identified. For example, in a very recent study, Champion et al 48 investigated secreted protein from intact biosafety level 2 pathogenic mycobacterial colonies using a modified version of whole-colony MALDI-time-of-flight (TOF)-MS.…”