HPLC‐ESI‐MS and MS/MS structural characterization of multifucosylated N‐glycoforms of the basic proline‐rich protein IB‐8a CON1⁺ in human saliva

Abstract

This study describes the characterization of the glycan moieties and the peptide backbone of six glycoforms of IB-8a CON1(+), a basic proline-rich protein present in human saliva. MS analyses on the intact glycoproteins before and after N-deglycosylation with PNGase F and high-resolution MS/MS sequencing by LTQ Orbitrap XL of peptides and glycopeptides from tryptic digests allowed the structural characterization of the glycan moieties and the polypeptide backbone, as well as to establish the glycosylation site at the asparagine residue at 98th position. Five of the glycoforms carry a biantennary N-linked glycan fucosylated in the innermost N-acetylglucosamine of the core and showing from zero to four additional fucoses in the antennal region. The sixth glycoform carries a monoantennary monofucosylated oligosaccharide. The glycoform cluster was detected on 28 of 71 adult saliva specimens. Level of fucosylation showed interindividual variability with the major relative abundance for the trifucosylated glycoform. Nonglycosylated IB-8a CON1(+) and the variant IB-8a CON1(-), lacking of the glycosylation site, have been also detected in human saliva.

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

“…This study at first confirms that high‐resolution ESI‐MS/MS carried out on intact protein (top‐down strategy) can characterize subtle modifications present in the protein primary structure . With regard to aPRPs polymorphisms, the possible allelic combinations of the most common aPRP isoforms generate 18 different phenotypes.…”

Separation methods hyphenated to mass spectrometry for the characterization of the protein glycosylation at the intact level

“…With regard to diagnosis, another example is the RPLC-MS analysis of 6 multifucosylated glycoforms of the basic proline-rich protein IB-8a CON1 + (so called according to its interaction with concanavalin A) in human saliva [89]. The sample pretreatment consisted of a simple dilution (1/1) with an acidic solution (0.2% TFA) and a centrifugation step for 10 min.…”

Salivary Peptidomics Targeting Clinical Applications

“…For instance, spectra manual inspection of tyrosine sulfation, a widespread PTM implicated in the intracellular trafficking of secreted peptides but often mismatched as phosphorylation (same neutral loss of 80 Da), resulted in the identification of four sulfated tyrosines (Tyr 27, 30, 34, and 36) in histatin 1 (18). Recently, the structure of six glycoforms of IB-8a was characterized, evidencing the presence of biantennary N-linked glycan fucosylated in the innermost N-acetylglucosamine of the core and showing from zero to four additional fucoses in the antenna (39). De novo sequencing also allowed the detection of N-and O-glycosylation sites in basic PRPs (38).…”