Identification of N-Linked Glycoproteins in Human Saliva by Glycoprotein Capture and Mass Spectrometry

Abstract

Glycoproteins make up a major and important part of the salivary proteome and play a vital role in maintaining the health of the oral cavity. Because changes in the physiological state of a person are reflected as changes in the glycoproteome composition, mapping the salivary glycoproteome will provide insights into various processes in the body. Salivary glycoproteins were identified by the hydrazide coupling and release method. In this approach, glycoproteins were coupled onto a hydrazide resin, the proteins were then digested and formerly N-glycosylated peptides were selectively released with the enzyme PNGase F and analyzed by LC-MS/MS. Employing this method, coupled with in-solution isoelectric focusing separation as an additional means for prefractionation, we identified 84 formerly N-glycosylated peptides from 45 unique N-glycoproteins. Of these, 16 glycoproteins have not been reported previously in saliva.In addition, we identified 44 new sites of N-linked glycosylation on the proteins.

Characterisation and expression of SPLUNC2, the human orthologue of rodent parotid secretory protein

“…This low level of sequence identity coupled with the (Geetha et al 2005). The protein has also been identified in whole saliva by a number of independent proteomic studies (Vitorino et al 2004;Ramachandran et al 2006;Gou et al 2006;Walz et al 2006) as well as in minor gland saliva (Siqueira et al 2008) and as a component of acquired pellicle (Siqueira et al 2007). Consistent with our proposal that PLUNCs may function in a host defence capacity we have shown that peptides derived from SPLUNC2 are able to inhibit LPS mediated cytokine release and also inhibit LBP/LPS interactions (Geetha et al 2005).…”

“…We have also shown that the protein is secreted into major gland saliva. Previously SPLUNC2 has not been identified in saliva by western analysis but has been identified in a number of proteomic studies of whole saliva, major gland saliva and, most recently, minor gland saliva (Vitorino et al 2004;Ramachandran et al 2006;Guo et al 2006;Walz et al 2006;Siqueira et al 2008). Our western analysis clearly shows that multiple protein isoforms of SPLUNC2 exist in saliva and that these are most likely due to differential glycosylation.…”

“…Our western analysis clearly shows that multiple protein isoforms of SPLUNC2 exist in saliva and that these are most likely due to differential glycosylation. This observation is supported by a proteomic study of glycosylated proteins in whole saliva (Ramachandran et al 2006). What function this differential glycosylation of SPLUNC2/psp serves, however, remains unclear.…”

“…We believe that this represents differences in the glycosylation status of the proteins within these tissues and suggest that tissue specific posttranslational modifications of SPLUNC2 may on occasion mask the epitope to which the antibody binds. We do note, however, that the two N-glycosylation sites identified in a proteomic analysis of saliva are not within the peptide epitopes chosen for antibody generation (Ramachandran et al 2006).…”

Solid-phase extraction of N-linked glycopeptides

“…SPEG has been successfully applied to the quantitative analysis of glycopeptides from complex mixtures, including serum/plasma and other body fluids 5,6,[11][12][13][14][15] , the detection of glycoproteins secreted or shed into the culture medium by cells, the detection of membrane proteins and cell-surface proteins from cells and tissues 16,17 and for comparing glycoproteins in the extracellular matrix of normal and disease tissues 18 . Several high-abundance plasma proteins including the most abundant plasma protein, albumin, do not appear to contain any N-glycosites and are therefore transparent to the method, allowing for more efficient identification and quantification of the lower abundance glycoproteins in blood 6 .…”