Large-scale identification of proteins in human salivary proteome by liquid chromatography/mass spectrometry and two-dimensional gel electrophoresis-mass spectrometry

Abstract

Human saliva contains a large number of proteins and peptides (salivary proteome) that help maintain homeostasis in the oral cavity. Global analysis of human salivary proteome is important for understanding oral health and disease pathogenesis. In this study, large-scale identification of salivary proteins was demonstrated by using shotgun proteomics and two-dimensinal gel electrophoresis-mass spectrometry (2-DE-MS). For the shotgun approach, whole saliva proteins were prefractionated according to molecular weight. The smallest fraction, presumably containing salivary peptides, was directly separated by capillary liquid chromatography (LC). However, the large protein fractions were digested into peptides for subsequent LC separation. Separated peptides were analyzed by on-line electrospray tandem mass spectrometry (MS/MS) using a quadrupole-time of flight mass spectrometer, and the obtained spectra were automatically processed to search human protein sequence database for protein identification. Additionally, 2-DE was used to map out the proteins in whole saliva. Protein spots 105 in number were excised and in-gel digested; and the resulting peptide fragments were measured by matrix-assisted laser desorption/ionization-mass spectrometry and sequenced by LC-MS/MS for protein identification. In total, we cataloged 309 proteins from human whole saliva by using these two proteomic approaches.

Complexity of the human whole saliva proteome

“…On this point of view, our results amplify the most recent saliva proteomic characterization (7,9,20) and do not support the previous conclusion that major proteins, such as α-amylase and albumin, display little if any modification (21).…”

“…In conclusion, with respect to the contradiction recently raised by the first proteomic characterizations of saliva (7,9,20,22), our data identify several proteins previously not reported in saliva and argue in favour of a complex proteome. This complexity appears to result from two opposite features: a relatively limited diversity of observed accessions, but counterbalanced by a large redundancy of corresponding spots.…”

“…Since protease inhibitors were added during the collection and storage, it should be emphasized that the possible degradations observed for α-amylase pattern could occur in the oral cavity, despite the abundance of protease inhibitors in saliva (cystatins, clade B; ca. one quarter of identified spots; 7,9,21,22). Perhaps the most intriguing situation corresponds to spots that possess peptides in both the N-terminal and Cterminal regions, but display MW on gel that is 25 % to 50 % lower than expected, suggesting the occurrence of internal deletions.…”

“…Saliva is usually taken as a complex medium, that was recently characterized by proteomic approach (7,9,20,21), allowing the cumulated identification of up to 35 accessions.…”

“…A main feature raising from the 320 protein spots that were characterized in these works (7,9,20,21), is that the saliva proteome contains a relatively low number of different accessions, but, conversely, spread out over a large number of protein spots (Table II).…”