Normal human saliva proteome related factors (gender and acid stimulation)
Abstract
As an easily available and noninvasive biological fluid, saliva is an attractive source for biomarker discovery. To date, more than 2000 proteins have been identified in human saliva and many studies have attempted to discover disease biomarkers in human saliva. However, little is known about specific changes in protein patterns by the influence of gender and acid stimulation. In this article, we analyzed the influence of gender and acid stimulation on normal human salivary proteome. A total of 1770 proteins were identified, and 82 proteins in unstimulated saliva were found to be gender specific, while 182 and 307 differential proteins were found to be acid stimulation specific in male samples and female samples, respectively. The results also showed acid stimulation caused more significant alteration and played a more important role in the human salivary proteome than gender. Therefore, when collecting saliva samples for disease biomarker research, it is necessary to consider the influence of acid stimulation and the factor of gender should be also taken into account.
Unstimulated and acid-stimulated saliva samples from 5 males and 5 females were labeled with 4-plex iTRAQ and analyzed by 2-DLC MS/MS. By bioinformatics analysis the gender and acid stimulation related proteins were defined. According to protein annotation the important proteins were validated by multiple reaction monitor analysis.
Experimental group
| No. | Gender | Age (year) | Concentration un-stimulated (ug/ul) | Concentration acid-stimulated (ug/ul) |
|---|---|---|---|---|
| 1 | female | 22 | 0.795 | 0.464 |
| 2 | female | 25 | 0.653 | 0.700 |
| 3 | female | 27 | 0.935 | 1.519 |
| 4 | female | 25 | 1.205 | 0.812 |
| 5 | female | 24 | 0.925 | 1.409 |
| 6 | male | 24 | 1.239 | 1.042 |
| 7 | male | 25 | 0.589 | 0.29 |
| 8 | male | 29 | 0.914 | 0.55 |
| 9 | male | 27 | 0.525 | 1.034 |
| 10 | male | 24 | 1.942 | 1.111 |
Validation group
| No. | Gender | Age (year) | Concentration un-stimulated (ug/ul) | Concentration acid-stimulated (ug/ul) |
|---|---|---|---|---|
| 11 | female | 22 | 0.563 | 0.374 |
| 12 | female | 25 | 0.992 | 0.479 |
| 13 | female | 27 | 1.345 | 0.432 |
| 14 | female | 25 | 0.553 | 0.809 |
| 15 | female | 24 | 1.03 | 0.511 |
| 16 | male | 24 | 0.812 | 0.894 |
| 17 | male | 25 | 0.771 | 0.667 |
| 18 | male | 29 | 0.884 | 1.502 |
| 19 | male | 27 | 0.562 | 0.822 |
| 20 | male | 24 | 0.383 | 0.75 |
| Protocol | Description |
|---|---|
| Clinical materials |
Unstimulated and acidstimulated saliva was collected from individuals. |
| Sample preparation |
All samples were centrifuged for 10 min at 14000×g, 4℃ to remove epithelial cells and other insoluble materials. Supernatants were transferred to a new tube on ice. After aliquoting, samples were stored at −80℃ for further analyses.The saliva protein concentration was determined using the Bradford protein assay. DTT was added to the samples to a final concentration of 20mM and iodoacetamide to a final concentration of 50 mM. Next, 8 M urea and 25 mM NH4HCO3 were used to wash the samples. The samples were digested overnight at 37℃ in 25mM NH4HCO3 containing trypsin at an enzyme/substrate ratio of 1:50. The peptides were desalted using HLB 1cc extraction cartridges (Oasis, Waters, Ireland). |
| iTRAQ Or TMT labeling |
The resulting peptides were labeled with the iTRAQ regent. Four samples (MUS, FUS, MAS, and FAS) were labeled by 113, 114, 115, and 116 iTRAQ 4-plex regents (AB Sciex, MA, USA), respectively, according to the manufacturer’s protocol.An equal amount ofall four labeled samples were combined into one sample and lyophilized. |
| HPLC |
The pooled mixture of labeled samples was fractionated using a high-pH RPLC column from Waters (4.6 × 250 mm, Xbridge C18, 3 um). The samples were loaded onto the column in buffer A2 (H2O, pH 10). The elution gradient was 5–30% buffer B2 (90% ACN, pH 10; flow rate = 1mL/min) for 60 min. The eluted peptides were collected at a rate of one fraction per minute, and the 60 dried fractions were resuspended in 0.1% formic acid and pooled into 20 samples by combining fractions 1, 11, 21; 2, 12, 22; 3, 13, 23, and so on. A total of 20 fractions from the saliva peptide mixtures (MUS, FUS, MAS, and FAS) were analyzed by LC–MS/MS. Each fraction was run twice. |
| LC/MS/MS |
Each fraction was analyzed using a reverse-phase C18 selfpacked capillary LC column (75 um × 100 mm, 3 um). The elution gradient was 5–30% buffer B1 (0.1% formic acid, 99.9% ACN; flow rate, 0.3 L/min) for 40 min. An Orbitrap Fusion Lumos mass spectrometer was used to analyze the eluted peptides, and each fraction was run two times. MS data were acquired in high-sensitivity mode using the following parameters: data-dependent MS/MS scans per full scan with top-speed mode (3 s), full scans acquired at a resolution of 60 000 and MS/MS scans at a resolution of 15000, 38% HCD collision energy, charge-state screening (including precursors with a charge state of +2 to +6), dynamic exclusion (exclusion duration 30 s), and a maximum injection time of 60 ms. |
| Data processing |
Proteome Discover (Thermo Fisher, Waltham, MA,USA; version 2.1) was used for database searching of the MS/MS results and was compared to the SwissProt human database (20 227 entries) assuming the use of trypsin. |