Normal human saliva proteome by 2DLC label-free approach
Body fluid is considered a rich source of disease biomarkers. Proteins in saliva have potential clinical applications for disease diagnostic and prognostic predictions. To determine the protein components and functional features, a proteomic analysis of saliva was conducted by high-resolution mass spectrometry. As a result, a total of 1771 proteins were identified, and the concentrations of 1265 proteins were estimated by an intensity-based algorithm quantitation method. Saliva is found in the mouth and sometimes is exposed to the outer environment; thus, infectious diseases can be spread through saliva. Many anti-inflammatory cells, including T helper cells, B cells, macrophages, monocytes, and natural killer cells, showed responses that were potentiated by saliva-enriched proteins.
In this study, we conducted a proteomic analysis using high-resolution mass spectrometry. For saliva, the relative protein abundances and average protein concentrations were estimated using intensity-based algorithm quantitation (iBAQ) methods.
All samples were obtained from normal healthy individuals. The volunteers were examined by physicians at the Peking Union Medical College Hospital.
Saliva is obtained from the Peking Union Medical College Hospital.
Pooled saliva samples were centrifuged to remove precipitates. The pooled saliva were depleted of 14 high-abundance proteins by using a Human 14 affinity LC column. The proteins were digested using filter-aided sample preparation methods.Briefly, the samples were denatured with 50 mM dithiothreitol, alkylated in 55 mM iodoacetamide, and digested with trypsin (1:50) at 37 °C overnight.
|iTRAQ Or TMT labeling|
After digestion, the lyophilized samples were fractionated by offline high pH reversed-phase liquid chromatography (RPLC) columns (4.6mm×250mm, C18, 3μm;Waters Corp,Milford,MA, USA).
Each sample was analyzed on a reverse-phase C18 self-packed capillary LC column (75 μm×100mm,3μm). A Triple TOF 5600 system was used to analyze the sample, and the MS data were acquired in a high-sensitivity mode using the following parameters: 30 data-dependent MS/MS scans per full scan; full scans were acquired at a resolution of 40 000, and MS/MS scans were acquired at a resolution of 20 000; rolling collision energy; charge state screening (including precursors with +2 to +4 charge states); dynamic exclusion (exclusion duration 15 s); MS/MS scan range of 250–1800 m/z; and a scan time of 50 ms.
The MS/MS data were processed using the Mascot software (version 2.3.02, Matrix Science, London, UK) and searched against the Swiss-Prot database (Homo sapiens, 20 267 sequences, 2013 07 version). The Mascot search results were filtered using the decoy database method in Scaffold (version 4.3.2, Proteome Software Inc., Portland, OR, USA).