Abstract
Saliva contains numerous proteins and peptides, each of them carries a number of biological functions that are very important in maintaining the oral cavity health and also yields information about both local and systemic diseases. Currently, proteomic analysis is the basis for large-scale identification of these proteins and discovery of new biomarkers for distinct diseases.
Objective
This study compared methodologies to extract salivary proteins for proteomic analysis.
Material and Methods
Saliva samples were collected from 10 healthy volunteers. In the first test, the necessity for using an albumin and IgG depletion column was evaluated, employing pooled samples from the 10 volunteers. In the second test, the analysis of the pooled samples was compared with individual analysis of one sample. Salivary proteins were extracted and processed for analysis by LC-ESI-MS/MS.
Results
In the first test, we identified only 35 proteins using the albumin and IgG depletion column, while we identified 248 proteins without using the column. In the second test, the pooled sample identified 212 proteins, such as carbonic anhydrase 6, cystatin isoforms, histatins 1 and 3, lysozyme C, mucin 7, protein S100A8 and S100A9, and statherin, while individual analysis identified 239 proteins, among which are carbonic anhydrase 6, cystatin isoforms, histatin 1 and 3, lactotransferrin, lyzozyme C, mucin 7, protein S100A8 and S100A9, serotransferrin, and statherin.
Conclusions
The standardization of protocol for salivary proteomic analysis was satisfactory, since the identification detected typical salivary proteins, among others. The results indicate that using the column for depletion of albumin and IgG is not necessary and that performing individual analysis of saliva samples is possible.
Keywords: Methods, Proteomics, Standardization, Saliva
Introduction
Saliva is a biological fluid composed of more than 99% water and less than 1% protein, electrolytes and other low-molecular-weight components. It originates mainly from three pairs of major salivary glands (parotid, submandibular and sublingual glands), as well as from 300 to 400 minor salivary glands present in the oral cavity. Saliva plays a key role in lubrication, chewing, swallowing and digestion. It protects the oral tissues and also provides biomarkers for local and systemic diseases 17 . Therefore, saliva contains more than 2000 proteins and peptides that are involved in an infinity of different biological functions in the oral cavity 17 . Saliva still plays a large role in the formation of acquired pellicle, which begins only a few seconds after exposure of the enamel to saliva 5 .
Human saliva is a biological fluid with enormous diagnostic potential. Because saliva can be noninvasively collected, it provides an attractive alternative for blood, serum or plasma 13 .
In the human saliva were identified 1166 proteins, and high portions of these proteins were found in serum. Currently, progress in salivary diagnostics has demonstrated that these contents can be very informative for detection of oral and systematic diseases 20 .
Proteomics, a new field of research centered on identification, quantitation, and characterization of proteins and their interplay, is largely based on the robustness, sensitivity, speed, and throughput of mass spectrometric procedures 6 . Currently, mass spectrometry is the basic technology for large-scale identification of these salivary proteins, and proteomic analysis of saliva has distinct advantages over blood, especially for proteins of low abundance 17 , 18 . One of the main challenges in proteomic analysis is the fact that highly abundant proteins can impair the identification of low-abundance proteins, considering the equipment dynamic range. In the case of saliva, albumin and immunoglobulin G (IgG), they are very abundant, and some authors have recommended using columns for depletion of these proteins during the extraction procedure 7 , 8 . Saliva functions are not only restricted to process food for digestion, considering that it contains a large number of proteins, which play important roles in the regulation of the immune defense and endocrine system and in the maintenance of mucosal tissue and dental health 1 .
Saliva may contain locally expressed proteins and other substances called biomarkers, which can be used as diseases' indicators, be closely related to an individual's health condition and change greatly when diseases occur. In general, most studies view saliva wrongly as a homogeneous body fluid. It is also not stable, but constantly in change, and its composition is affected among other things by sampling methodology, environment, periodicity, oral hygiene, psychological status and general health 6 , 13 , 20 .
Considering the importance of saliva in the oral cavity homeostasis, as well as its great potential as a diagnostic fluid, the aim of this study was to standardize a protocol to extract salivary proteins for further proteomic analysis. In the first test, we evaluated the need for using an albumin and IgG column to deplete these proteins during protein extraction. In the second test, we compared analysis of samples pooled from 10 volunteers with samples from individual analysis.
Material and methods
Ethical aspects and human subjects
The protocol of this study was submitted and approved by the Ethics Committee in Research with Human Beings of the Bauru School of Dentistry - FOB/USP (CAAE No. 61484116.0.0000.5417). Ten participants with good general and oral health took part of this study, which was based on previous in vivo studies 18 . Inclusion criteria were: nonsmokers with good general and oral health, stimulated salivary flow >1 mL/min and unstimulated salivary flow >0.25 mL/min, salivary pH>6.0.
Saliva collection
The volunteers were asked to rest for 15 min before collecting saliva, sitting upright. They were asked not to speak or eat before beginning to collect saliva. First, they rinsed their mouths with 5 mL of drinking deionized water, then they were asked to swallow saliva for 5 min. After this period, the volunteers spit out all the saliva accumulated in the mouth in a plastic tube immersed in ice for 10 min (unstimulated flow). The saliva samples were immediately centrifuged at 14,000 g for 15 min at 4°C to remove all debris, such as insoluble material, cell debris and food debris. The supernatant from each sample was collected and frozen at -80°C until analysis. These procedures were based on previous studies 6 , 18 .
Preparation of the saliva samples
The experiments were performed into two phases. The first test was done to evaluate whether or not the albumin & IgG Depletion SpinTrap column (GE Healthcare®, Buckinghamshire, UK) should be used. The second test was performed after the results of the first to compare analysis of salivary samples pooled from all the 10 volunteers with analysis of an individual sample from one selected volunteer.
For the first test, 100 μl of saliva from each volunteer was taken and transferred to 10 new tubes. For the second test, 100 μl of each saliva sample was also taken and transferred to 10 new tubes to constitute the pool, while 1 ml of saliva was taken from only one of the volunteers (randomly selected) for individual analysis.
Proteins from the saliva samples were extracted using an equal volume of a solution containing 6 M urea, 2 thiourea in 50 mM NH4HCO3 pH 7.8. The samples were vortexed at 4°C for 10 min, sonicated for 5 min and centrifuged at 14,000 g at 4°C for 10 min. This step was repeated once more. For the first test (with or without the use of the albumin and IgG depletion column), we added 100 μl of the extraction solution to each Eppendorf tube. For the second test (pool X individual analysis), we added 100 µl of the extraction solution in each Eppendorf tube (for the samples that will be pooled later on), while for the individual sample, we added 1 ml of the extraction solution. In all the cases, an equal volume of saliva sample and extraction solution was used. For the pooled samples, we placed the content of the 10 tubes in one tube after the extraction procedure, constituting the pool for further analysis.
After extraction, for the first test, the pooled sample was loaded into the albumin & IgG depletion columns, according to the manufacturer´s instructions Albumin & IgG Depletion SpinTrap column (GE Healthcare®, Buckinghamshire, UK). We did not use this column in the second test.
The samples were then concentrated to 150 μl in Falcon Amicon tubes (Merck Millipore®, Tullagreen, County Cork, Ireland). After concentration, the samples were reduced with 5 mM dithiothreitol (DTT) for 40 min at 37°C, alkylated with 10 mM iodoacetamide (IAA) for 30 min in the dark. After this procedure, we added 100 μl of 50 mM NH4HCO3, and the samples were digested with 2% (w/w) trypsin (Promega®, Madison, USA) for 14 hours at 37°C. After this period, we added 10 µl of 5% formic acid to stop the trypsin reaction, then the samples were purified and desalted using the C18 Spin columns (Thermo Scientific®, Rockford, Illinois, USA) and we withdrew a 1 ul aliquot of each sample from the tests for protein quantification by the Bradford method (Bio-Rad®, Hercules, Califórnia, USA) 16 . We resuspended the samples in the solution containing 3% acetonitrile and 0.1% formic acid to be submitted to Nano Liquid Chromatography Electron Spray Ionization Tandem Mass Spectrometry - LC-ESI-MS/MS (Waters, Manchester, New Hampshire, UK).
Shotgun label-free quantitative proteomic analysis
Peptides identification was performed on a nanoACQUITY UPLC-Xevo QTof MS system (Waters, Manchester, New Hampshire, UK). The nanoACQUITY UPLC was equipped with nanoACQUITY HSS T3, analytical reverse phase column (75 μm X 150 mm, 1.8 μm particle size (Waters, Manchester, New Hampshire, UK). The column was equilibrated with mobile phase A (0.1% formic acid in water). Then, the peptides were separated with a linear gradient of 7-85% mobile phase B (0.1% formic acid in ACN) for 70 min at a flow rate of 0.35 μL/min. The column temperature was maintained at 55°C. The Xevo G2 Q-TOF mass spectrometer was operated in positive nano-electrospray ion mode, and data were collected using the MSE method in elevated energy (19-45 V), which allows data acquisition of both precursor and fragment ions, in one injection. Source conditions used included capillary voltage, 2.5 kV; sample cone, 30 V; extraction cone, 5.0 V and source temperature, 800C. Data acquisition occurred over 70 min, and the scan range was 50–2000 Da. The lockspray, used to ensure accuracy and reproducibility, was run with a [Glu1] fibrinopeptide solution (1 pmol/μL) at a flow rate of 1 μL/min, as a reference ion in positive mode at m/z 785.8427. ProteinLynx Global Server (PLGS) version 3.0 was used to process and search the LC-MSE continuum data. Proteins were identified with the embedded ion accounting algorithm in the software and a search of the Homo sapiens database (reviewed only, UniProtKB/Swiss-Prot) downloaded on September 2015 from UniProtKB (http://www.uniprot.org/). The use of human database excludes the identification of bacterial proteins that could be present in the saliva.
Results
In the first test, when the albumin and IgG depletion column was used, the total amount of protein recovered from the pooled samples after extraction was 8 μg, while only 35 salivary proteins were identified. Among them are proteins typically found in saliva, such as alpha-amylase 1 and 2B, cystatin isoforms, hemoglobin isoforms and mucin 7, among others (Table 1). When the depletion column was not used, the amount of protein recovered was much higher (48.0 μg) and 248 proteins were identified, among them many typical components of saliva such as alpha-amylase 1 and 2B, many cystatin isoforms, carbonic anhydrase 6, lactotransferrin, lysozyme C, mucin 7, proline-rich protein 4, protein S100A9, serotransferrin, statherin, several hemoglobin isoforms, among others (Table 2).
Table 1. Salivary proteins identified when the albumin and IgG depletion column was used.
| Accession number | Protein name | score | Cover (%) |
|---|---|---|---|
| P04745 | Alpha-amylase 1 | 7589.70 | 54.99 |
| P19961 | Alpha-amylase 2B | 6833.20 | 47.75 |
| P04280 | Basic salivary proline-rich protein 1 | 488.14 | 43.88 |
| P02812 | Basic salivary proline-rich protein 2 | 3642.44 | 45.67 |
| P49407 | Beta-arrestin-1 | 158.66 | 9.09 |
| P01036 | Cystatin-S | 1465.11 | 31.91 |
| P09228 | Cystatin-AS | 516.59 | 24.11 |
| P01037 | Cystatin-SN | 1378.19 | 21.28 |
| Q9UGM3 | Deleted in malignant brain tumors 1 protein | 98.93 | 2.11 |
| P14867 | Gamma-aminobutyric acid receptor subunit alpha-1 | 92.53 | 7.46 |
| G3V1N2 | HCG1745306_ isoform CRA_a | 456.20 | 22.73 |
| P69905 | Hemoglobin subunit alpha | 1306.87 | 28.17 |
| P68871 | Hemoglobin subunit beta | 1659.66 | 66.67 |
| P02042 | Hemoglobin subunit delta | 497.84 | 25.17 |
| A0A0G2JMB2 | Ig alpha-2 chain C region (Fragment) | 559.94 | 16.76 |
| P01876 | Immunoglobulin heavy constant alpha 1 | 912.82 | 30.59 |
| P01877 | Immunoglobulin heavy constant alpha 2 | 345.30 | 20.00 |
| P01591 | Immunoglobulin J chain | 1363.63 | 36.48 |
| P01834 | Immunoglobulin kappa constant | 333.71 | 51.40 |
| P0CG04 | Immunoglobulin lambda constant 1 | 136.40 | 14.15 |
| P0DOY2 | Immunoglobulin lambda constant 2 | 165.46 | 23.58 |
| P0DOY3 | Immunoglobulin lambda constant 3 | 153.74 | 23.58 |
| P0CF74 | Immunoglobulin lambda constant 6 | 136.40 | 14.15 |
| B9A064 | Immunoglobulin lambda-like polypeptide 5 | 136.40 | 7.01 |
| P31025 | Lipocalin-1 | 1181.01 | 26.70 |
| Q8TAX7 | Mucin-7 | 95.21 | 3.71 |
| P04746 | Pancreatic alpha-amylase | 6723.99 | 41.49 |
| P01833 | Polymeric immunoglobulin receptor | 305.15 | 15.58 |
| P12273 | Prolactin-inducible protein | 1027.80 | 40.41 |
| A0A0A0MT31 | Proline-rich protein 4 | 8108.76 | 72.29 |
| Q5VSP4 | Putative lipocalin 1-like protein 1 | 958.48 | 6.79 |
| P02810 | Salivary acidic proline-rich phosphoprotein 1/2 | 8108.76 | 72.29 |
| P02814 | Submaxillary gland androgen-regulated protein 3B | 2090.48 | 65.82 |
| A0A087WZY1 | Uncharacterized protein | 7158.08 | 16.60 |
| Q96DA0 | Zymogen granule protein 16 homolog B | 721.70 | 41.83 |
Standardization of a protocol for shotgun proteomic analysis of saliva
Table 2. Salivary proteins identified when the albumin and IgG depletion column was not used.
| Accession number | Protein name | score | Cover (%) |
|---|---|---|---|
| Q15118 | [Pyruvate dehydrogenase (acetyl-transferring)] kinase isozyme 1_ mitochondrial | 89.50 | 8.26 |
| P31946 | 14-3-3 protein beta/alpha | 166.37 | 3.25 |
| P62258 | 14-3-3 protein epsilon | 177.85 | 3.14 |
| Q04917 | 14-3-3 protein eta | 166.37 | 3.25 |
| P61981 | 14-3-3 protein gamma | 166.37 | 3.24 |
| P31947 | 14-3-3 protein sigma | 166.37 | 3.23 |
| P27348 | 14-3-3 protein theta | 195.23 | 12.65 |
| P63104 | 14-3-3 protein zeta/delta | 166.37 | 3.27 |
| Q6ZVK8 | 8-oxo-dGDP phosphatase NUDT18 | 138.11 | 19.50 |
| E5KP25 | A/G-specific adenine DNA glycosylase | 242.24 | 5.28 |
| P68032 | Actin_ alpha cardiac muscle 1 | 10751.18 | 40.05 |
| P68133 | Actin_ alpha skeletal muscle | 10681.87 | 33.95 |
| P62736 | Actin_ aortic smooth muscle | 10396.48 | 37.14 |
| P60709 | Actin_ cytoplasmic 1 | 18715.02 | 66.67 |
| P63261 | Actin_ cytoplasmic 2 | 18715.02 | 66.67 |
| P63267 | Actin_ gamma-enteric smooth muscle | 10327.17 | 31.12 |
| Q6P461 | Acyl-coenzyme A synthetase ACSM6_ mitochondrial | 399.16 | 13.33 |
| Q9UIF7 | Adenine DNA glycosylase | 242.24 | 5.31 |
| Q9Y6U3 | Adseverin | 51.66 | 5.17 |
| C9JKR2 | Albumin_ isoform CRA_k | 25004.47 | 77.94 |
| P02763 | Alpha-1-acid glycoprotein 1 | 259.49 | 7.46 |
| P01009 | Alpha-1-antitrypsin | 114.17 | 14.59 |
| P01023 | Alpha-2-macroglobulin | 195.37 | 14.25 |
| P04745 | Alpha-amylase 1 | 125762.3 | 77.69 |
| P19961 | Alpha-amylase 2B | 85518.55 | 67.91 |
| Q69YU3 | Ankyrin repeat domain-containing protein 34A | 213.80 | 23.19 |
| Q5T3N1 | Annexin (Fragment) | 419.03 | 34.31 |
| P04083 | Annexin A1 | 454.28 | 33.53 |
| P03973 | Antileukoproteinase | 822.96 | 40.15 |
| Q16671 | Anti-Muellerian hormone type-2 receptor | 646.30 | 18.32 |
| P02647 | Apolipoprotein A-I | 436.68 | 32.58 |
| B1APP8 | ATP-dependent 6-phosphofructokinase_ platelet type | 156.72 | 21.29 |
| O14965 | Aurora kinase A | 187.17 | 8.93 |
| P04280 | Basic salivary proline-rich protein 1 | 13742.73 | 44.39 |
| P02812 | Basic salivary proline-rich protein 2 | 36329.24 | 69.23 |
| Q6W2J9 | BCL-6 corepressor | 171.50 | 2.34 |
| P61769 | Beta-2-microglobulin | 7681.87 | 54.62 |
| Q562R1 | Beta-actin-like protein 2 | 1631.58 | 17.02 |
| Q96DR5 | BPI fold-containing family A member 2 | 4054.46 | 40.56 |
| Q8TDL5 | BPI fold-containing family B member 1 | 238.42 | 27.27 |
| Q8N4F0 | BPI fold-containing family B member 2 | 4941.71 | 32.97 |
| Q8N4G4 | CA6 protein | 236.85 | 4.47 |
| P23280 | Carbonic anhydrase 6 | 1927.33 | 43.83 |
| P07339 | Cathepsin D | 153.05 | 17.96 |
| H0YDT2 | Cathepsin W (Fragment) | 152.45 | 12.32 |
| A0A087X2B6 | Cell cycle and apoptosis regulator protein 2 | 186.22 | 13.60 |
| O60308 | Centrosomal protein of 104 kDa | 36.50 | 3.35 |
| O94986 | Centrosomal protein of 152 kDa | 24.18 | 5.03 |
| O75153 | Clustered mitochondria protein homolog | 864.26 | 9.93 |
| P35606 | Coatomer subunit beta' | 186.05 | 6.73 |
| G3V1A4 | Cofilin 1 (Non-muscle)_ isoform CRA_a | 613.65 | 18.79 |
| P23528 | Cofilin-1 | 613.65 | 16.87 |
| Q8TD31 | Coiled-coil alpha-helical rod protein 1 | 47.65 | 2.43 |
| Q9P0B6 | Coiled-coil domain-containing protein 167 | 170.32 | 15.46 |
| P01024 | Complement C3 | 181.96 | 9.32 |
| Q2VPA4 | Complement component receptor 1-like protein | 148.59 | 7.21 |
| P04080 | Cystatin-B | 3144.06 | 55.10 |
| P01034 | Cystatin-C | 1547.12 | 31.51 |
| P28325 | Cystatin-D | 535.37 | 47.89 |
| P01036 | Cystatin-S | 41046.83 | 73.76 |
| P09228 | Cystatin-SA | 21107.61 | 53.90 |
| P01037 | Cystatin-SN | 40764.24 | 68.09 |
| P54108 | Cysteine-rich secretory protein 3 | 371.45 | 26.94 |
| Q9UGM3 | Deleted in malignant brain tumors 1 protein | 274.04 | 6.80 |
| Q8IYB7 | DIS3-like exonuclease 2 | 192.96 | 5.42 |
| Q9NVU0 | DNA-directed RNA polymerase III subunit RPC5 | 187.74 | 4.66 |
| Q1HG43 | Dual oxidase maturation factor 1 | 248.89 | 13.12 |
| O95714 | E3 ubiquitin-protein ligase HERC2 | 190.34 | 5.05 |
| Q8NG27 | E3 ubiquitin-protein ligase Praja-1 | 680.83 | 14.31 |
| P43897 | Elongation factor Ts_ mitochondrial | 129.02 | 9.23 |
| Q0PNE2 | Elongator complex protein 6 | 63.64 | 13.53 |
| V9HW75 | Epididymis secretory protein Li 109 | 337.33 | 22.86 |
| P02675 | Fibrinogen beta chain | 420.77 | 40.73 |
| P02679 | Fibrinogen gamma chain | 453.82 | 22.52 |
| Q0PRL4 | Forkhead box P2 variant 3 | 142.49 | 10.19 |
| Q8N6B5 | Forkhead box P2_ isoform CRA_d (Fragment) | 142.49 | 11.84 |
| O15409 | Forkhead box protein P2 | 199.65 | 12.45 |
| O95872 | G patch domain and ankyrin repeat-containing protein 1 | 268.32 | 17.70 |
| P19526 | Galactoside 2-alpha-L-fucosyltransferase 1 | 174.70 | 13.42 |
| P48058 | Glutamate receptor 4 | 50.22 | 2.55 |
| P04406 | Glyceraldehyde-3-phosphate dehydrogenase | 190.90 | 16.72 |
| P00738 | Haptoglobin | 349.21 | 24.88 |
| G3V1N2 | HCG1745306_ isoform CRA_a | 22783.57 | 58.18 |
| P69905 | Hemoglobin subunit alpha | 27452.86 | 59.15 |
| P68871 | Hemoglobin subunit beta | 49667.26 | 95.24 |
| P02042 | Hemoglobin subunit delta | 9498.60 | 33.33 |
| P02100 | Hemoglobin subunit epsilon | 1940.46 | 6.80 |
| P69891 | Hemoglobin subunit gamma-1 | 1940.46 | 6.80 |
| P69892 | Hemoglobin subunit gamma-2 | 1940.46 | 6.80 |
| P02790 | Hemopexin | 460.96 | 22.51 |
| P15515 | Histatin-1 | 32092.25 | 36.84 |
| P15516 | Histatin-3 | 7558.25 | 13.73 |
| P57058 | Hormonally up-regulated neu tumor-associated kinase | 218.10 | 3.50 |
| Q9BS19 | HPX protein | 352.10 | 21.65 |
| A0A0G2JMB2 | Ig alpha-2 chain C region (Fragment) | 22147.53 | 68.24 |
| A0A0A0MS07 | Ig gamma-1 chain C region (Fragment) | 1490.66 | 45.76 |
| A0A087WYJ9 | Ig mu chain C region | 2129.91 | 40.71 |
| P04220 | Ig mu heavy chain disease protein | 1800.88 | 31.97 |
| P01876 | Immunoglobulin heavy constant alpha 1 | 25196.43 | 61.19 |
| P01877 | Immunoglobulin heavy constant alpha 2 | 18459.82 | 64.12 |
| P01857 | Immunoglobulin heavy constant gamma 1 | 3671.28 | 50.91 |
| P01859 | Immunoglobulin heavy constant gamma 2 | 729.35 | 38.34 |
| P01860 | Immunoglobulin heavy constant gamma 3 | 487.81 | 24.93 |
| P01861 | Immunoglobulin heavy constant gamma 4 | 599.47 | 20.18 |
| P01871 | Immunoglobulin heavy constant mu | 2171.72 | 47.68 |
| A0A075B7F0 | Immunoglobulin heavy variable 3/OR16-10 (non-functional) (Fragment) | 378.41 | 9.48 |
| S4R460 | Immunoglobulin heavy variable 3/OR16-9 (non-functional) | 5403.28 | 31.25 |
| P01762 | Immunoglobulin heavy variable 3-11 | 378.41 | 9.40 |
| P01766 | Immunoglobulin heavy variable 3-13 | 378.41 | 9.48 |
| A0A0C4DH32 | Immunoglobulin heavy variable 3-20 (Fragment) | 378.41 | 9.40 |
| A0A0B4J1V1 | Immunoglobulin heavy variable 3-21 | 378.41 | 9.40 |
| A0A0B4J1X8 | Immunoglobulin heavy variable 3-43 | 378.41 | 9.32 |
| P01763 | Immunoglobulin heavy variable 3-48 | 378.41 | 9.40 |
| P01780 | Immunoglobulin heavy variable 3-7 | 401.30 | 17.09 |
| P01782 | Immunoglobulin heavy variable 3-9 | 378.41 | 9.32 |
| P01591 | Immunoglobulin J chain | 18415.28 | 42.14 |
| P01834 | Immunoglobulin kappa constant | 16816.83 | 85.98 |
| P0CG04 | Immunoglobulin lambda constant 1 | 9338.45 | 77.36 |
| P0DOY2 | Immunoglobulin lambda constant 2 | 13921.14 | 77.36 |
| P0DOY3 | Immunoglobulin lambda constant 3 | 13921.14 | 77.36 |
| P0CF74 | Immunoglobulin lambda constant 6 | 13267.04 | 50.94 |
| A0M8Q6 | Immunoglobulin lambda constant 7 | 10499.89 | 36.79 |
| B9A064 | Immunoglobulin lambda-like polypeptide 5 | 9338.45 | 38.32 |
| P08069 | Insulin-like growth factor 1 receptor | 32.75 | 5.63 |
| P06870 | Kallikrein-1 | 227.71 | 10.31 |
| Q9Y5K2 | Kallikrein-4 | 304.56 | 17.72 |
| P13645 | Keratin_ type I cytoskeletal 10 | 297.80 | 2.05 |
| Q99456 | Keratin_ type I cytoskeletal 12 | 421.18 | 14.17 |
| P13646 | Keratin_ type I cytoskeletal 13 | 4810.33 | 46.94 |
| P02533 | Keratin_ type I cytoskeletal 14 | 158.42 | 4.24 |
| P19012 | Keratin_ type I cytoskeletal 15 | 1164.86 | 14.25 |
| P08779 | Keratin_ type I cytoskeletal 16 | 158.42 | 4.23 |
| Q04695 | Keratin_ type I cytoskeletal 17 | 143.47 | 2.08 |
| P08727 | Keratin_ type I cytoskeletal 19 | 529.84 | 6.75 |
| P35908 | Keratin_ type II cytoskeletal 2 epidermal | 300.25 | 22.07 |
| Q01546 | Keratin_ type II cytoskeletal 2 oral | 165.14 | 12.07 |
| P19013 | Keratin_ type II cytoskeletal 4 | 876.71 | 42.13 |
| P13647 | Keratin_ type II cytoskeletal 5 | 489.99 | 7.97 |
| P02538 | Keratin_ type II cytoskeletal 6A | 794.78 | 31.56 |
| P04259 | Keratin_ type II cytoskeletal 6B | 765.88 | 28.01 |
| P48668 | Keratin_ type II cytoskeletal 6C | 765.88 | 28.01 |
| O95678 | Keratin_ type II cytoskeletal 75 | 190.38 | 3.81 |
| Q5XKE5 | Keratin_ type II cytoskeletal 79 | 190.38 | 3.93 |
| O14777 | Kinetochore protein NDC80 homolog | 410.89 | 9.03 |
| P22079 | Lactoperoxidase | 1724.32 | 34.13 |
| P02788 | Lactotransferrin | 382.65 | 32.11 |
| Q9C099 | Leucine-rich repeat and coiled-coil domain-containing protein 1 | 270.77 | 9.98 |
| Q9NPC1 | Leukotriene B4 receptor 2 | 209.15 | 4.37 |
| P31025 | Lipocalin-1 | 19334.38 | 57.95 |
| P28330 | Long-chain specific acyl-CoA dehydrogenase_ mitochondrial | 137.44 | 9.07 |
| Q8IYD9 | Lung adenoma susceptibility protein 2 | 141.09 | 9.14 |
| P61626 | Lysozyme C | 10190.75 | 70.27 |
| Q14680 | Maternal embryonic leucine zipper kinase | 208.24 | 8.14 |
| P42679 | Megakaryocyte-associated tyrosine-protein kinase | 156.39 | 10.85 |
| P01033 | Metalloproteinase inhibitor 1 | 858.61 | 44.44 |
| Q2QL34 | Mpv17-like protein | 240.73 | 11.73 |
| Q8TAX7 | Mucin-7 | 11686.20 | 15.65 |
| Q8NCY6 | Myb/SANT-like DNA-binding domain-containing protein 4 | 176.81 | 11.30 |
| P24158 | Myeloblastin | 175.85 | 4.69 |
| Q8NCE2 | Myotubularin-related protein 14 | 342.16 | 19.38 |
| Q9NYA4 | Myotubularin-related protein 4 | 234.57 | 15.82 |
| F8WCT3 | NEDD8-conjugating enzyme UBE2F | 167.98 | 37.18 |
| P59665 | Neutrophil defensin 1 | 1037.46 | 17.02 |
| P59666 | Neutrophil defensin 3 | 1037.46 | 17.02 |
| O00221 | NF-kappa-B inhibitor epsilon | 176.16 | 6.80 |
| Q2L696 | Nucb2 splice variant | 337.33 | 24.62 |
| Q14980 | Nuclear mitotic apparatus protein 1 | 278.08 | 4.96 |
| Q9Y618 | Nuclear receptor corepressor 2 | 44.62 | 3.33 |
| A0A087WSV8 | Nucleobindin 2_ isoform CRA_b | 337.33 | 22.86 |
| P80303 | Nucleobindin-2 | 337.33 | 22.86 |
| O75414 | Nucleoside diphosphate kinase 6 | 140.72 | 14.52 |
| C9JQB1 | Nucleoside diphosphate kinase | 140.72 | 19.15 |
| Q9GZK3 | Olfactory receptor 2B2 | 166.49 | 19.33 |
| Q5SZR7 | Ornithine decarboxylase antizyme 3 | 300.95 | 18.55 |
| Q7RTY7 | Ovochymase-1 | 190.59 | 10.05 |
| P04746 | Pancreatic alpha-amylase | 79860.79 | 59.10 |
| P13796 | Plastin-2 | 364.90 | 18.02 |
| P13797 | Plastin-3 | 259.13 | 4.29 |
| P01833 | Polymeric immunoglobulin receptor | 10715.77 | 41.62 |
| Q6S8J3 | POTE ankyrin domain family member E | 7556.27 | 11.07 |
| A5A3E0 | POTE ankyrin domain family member F | 7557.11 | 13.67 |
| P0CG38 | POTE ankyrin domain family member I | 6915.24 | 6.79 |
| P0CG39 | POTE ankyrin domain family member J | 2868.60 | 5.97 |
| P51531 | Probable global transcription activator SNF2L2 | 158.85 | 2.01 |
| Q53EL6 | Programmed cell death protein 4 | 138.40 | 8.74 |
| P12273 | Prolactin-inducible protein | 31682.10 | 76.71 |
| Q16378 | Proline-rich protein 4 | 312.60 | 21.64 |
| H0Y4B9 | Propionyl-CoA carboxylase alpha chain_ mitochondrial (Fragment) | 231.31 | 20.90 |
| P07602 | Prosaposin | 205.84 | 9.35 |
| D6RDZ2 | Protein FAM193B (Fragment) | 266.86 | 35.56 |
| Q14320 | Protein FAM50A | 176.55 | 10.62 |
| Q5VT40 | Protein FAM78B | 141.80 | 10.73 |
| Q8N7I0 | Protein GVQW1 | 164.91 | 17.95 |
| Q6P5S2 | Protein LEG1 homolog | 1162.24 | 29.09 |
| Q8ND56 | Protein LSM14 homolog A | 270.50 | 9.50 |
| Q8WYL5 | Protein phosphatase Slingshot homolog 1 | 322.72 | 3.91 |
| Q5THK1 | Protein PRR14L | 367.74 | 10.13 |
| P06702 | Protein S100-A9 | 571.65 | 39.47 |
| Q96EA4 | Protein Spindly | 138.75 | 2.64 |
| Q58EX7 | Puratrophin-1 | 166.93 | 2.60 |
| Q9BYX7 | Putative beta-actin-like protein 3 | 1002.92 | 10.67 |
| Q5VSP4 | Putative lipocalin 1-like protein 1 | 3906.17 | 11.11 |
| A8K554 | Putative protein ZNF815 | 163.67 | 26.15 |
| Q96GD0 | Pyridoxal phosphate phosphatase | 92.62 | 11.15 |
| H3BR70 | Pyruvate kinase | 336.60 | 18.03 |
| P14618 | Pyruvate kinase PKM | 336.60 | 12.43 |
| Q15276 | Rab GTPase-binding effector protein 1 | 349.01 | 8.24 |
| H3BPI9 | Receptor protein serine/threonine kinase (Fragment) | 641.71 | 47.67 |
| P02810 | Salivary acidic proline-rich phosphoprotein 1/2 | 40463.03 | 26.51 |
| Q14674 | Separin | 32.80 | 4.39 |
| Q9BZL6 | Serine/threonine-protein kinase D2 | 165.21 | 3.87 |
| B4DTS2 | Serine/threonine-protein kinase | 165.21 | 3.83 |
| J3QLP4 | Serine/threonine-protein kinase RIO3 (Fragment) | 335.03 | 50.56 |
| G3V5U8 | Serine/threonine-protein phosphatase 2A regulatory subunit B'' subunit gamma | 157.81 | 24.53 |
| P02787 | Serotransferrin | 5631.55 | 44.41 |
| P02768 | Serum albumin | 65771.62 | 81.28 |
| P40763 | Signal transducer and activator of transcription 3 | 43.20 | 6.10 |
| Q9UBC9 | Small proline-rich protein 3 | 424.01 | 65.09 |
| A1L4H1 | Soluble scavenger receptor cysteine-rich domain-containing protein SSC5D | 62.36 | 2.67 |
| P02808 | Statherin | 52769.28 | 53.23 |
| P02814 | Submaxillary gland androgen-regulated protein 3B | 52053.05 | 65.82 |
| Q9UMS6 | Synaptopodin-2 | 184.00 | 1.83 |
| G5E9B5 | TCF3 (E2A) fusion partner (In childhood Leukemia)_ isoform CRA_b | 165.61 | 19.67 |
| Q8WW35 | Tctex1 domain-containing protein 2 | 188.69 | 14.08 |
| Q7Z6L1 | Tectonin beta-propeller repeat-containing protein 1 | 350.11 | 12.62 |
| Q9UKR8 | Tetraspanin-16 | 313.97 | 27.35 |
| P20061 | Transcobalamin-1 | 230.38 | 20.32 |
| A6H8Y1 | Transcription factor TFIIIB component B'' homolog | 167.29 | 2.82 |
| O95359 | Transforming acidic coiled-coil-containing protein 2 | 372.27 | 6.41 |
| P55072 | Transitional endoplasmic reticulum ATPase | 236.03 | 10.92 |
| P29401 | Transketolase | 133.40 | 13.80 |
| Q8NDV7 | Trinucleotide repeat-containing gene 6A protein | 180.44 | 3.98 |
| K7EQY5 | Tyrosine-protein kinase | 156.39 | 10.87 |
| Q86TW2 | Uncharacterized aarF domain-containing protein kinase 1 | 174.76 | 10.57 |
| H3BMD7 | Uncharacterized protein (Fragment) | 240.73 | 19.49 |
| A0A087WZK3 | Uncharacterized protein (Fragment) | 469.24 | 43.09 |
| A0A087WZY1 | Uncharacterized protein | 40463.03 | 16.60 |
| A0A087WUV0 | Uncharacterized protein | 464.85 | 8.62 |
| E7ESA3 | Uncharacterized protein | 188.69 | 18.87 |
| Q9HB07 | UPF0160 protein MYG1_ mitochondrial | 435.46 | 12.23 |
| Q9NY84 | Vascular non-inflammatory molecule 3 | 540.71 | 10.58 |
| Q14508 | WAP four-disulfide core domain protein 2 | 1637.99 | 33.87 |
| E9PDB0 | WD repeat-containing protein 49 | 424.40 | 5.02 |
| Q86UP3 | Zinc finger homeobox protein 4 | 205.12 | 3.06 |
| Q5FWF6 | Zinc finger protein 789 | 138.52 | 9.41 |
| Q17R98 | Zinc finger protein 827 | 296.41 | 2.87 |
| P25311 | Zinc-alpha-2-glycoprotein | 5026.17 | 55.03 |
| Q96DA0 | Zymogen granule protein 16 homolog B | 47333.93 | 56.73 |
In the second test, for comparison of analysis of pooled versus individual sample, the depletion column was not used. For the pooled sample, the amount of protein recovered after extraction was 54.02 µg, which allowed the identification of 212 proteins, including alpha-amylase 1 and 2B, carbonic anhydrase 6, cystatin isoforms (B, C, D, S, SA, SN), histatin 1 and 3, lysozyme C, mucin 7, protein S100A8 and S100A9, statherin, several hemoglobin isoforms, among others (Table 3). In the analysis of the individual sample, 25.13 μg of total protein were obtained and 239 proteins were identified, among which are alpha-amylase 1 and 2B, alpha-enolase, carbonic anhydrase 6, many cystatin isoforms (B, C-D, S, SA, SN), histatin 1 and 3, Ig alpha-2 chain C region, Ig a chain C region, lactotransferrin, lysozyme C, mucin 7, protein S1008 and S100A9, serotransferrin, statherin, among other proteins (Table 4).
Table 3. Proteins of the saliva identified in the pool analysis.
| Accession number | Protein name | score | Cover(%) |
|---|---|---|---|
| P16885 | 1-phosphatidylinositol 4_5-bisphosphate phosphodiesterase gamma-2 | 314.78 | 4.51 |
| P68032 | Actin_ alpha cardiac muscle 1 | 6085.31 | 31.30 |
| P68133 | Actin_ alpha skeletal muscle | 6085.31 | 31.30 |
| P62736 | Actin_ aortic smooth muscle | 4676.94 | 28.38 |
| P60709 | Actin_ cytoplasmic 1 | 17496 | 67.20 |
| P63261 | Actin_ cytoplasmic 2 | 17496 | 67.20 |
| P63267 | Actin_ gamma-enteric smooth muscle | 4676.94 | 28.46 |
| Q01518 | Adenylyl cyclase-associated protein 1 | 440.27 | 26.11 |
| C9JKR2 | Albumin_ isoform CRA_k | 26466.72 | 74.82 |
| P01009 | Alpha-1-antitrypsin | 2252.60 | 22.97 |
| P01023 | Alpha-2-macroglobulin | 665.70 | 22.86 |
| P04745 | Alpha-amylase 1 | 153591.90 | 78.86 |
| P19961 | Alpha-amylase 2B | 110753.50 | 58.51 |
| P06733 | Alpha-enolase | 1637.76 | 33.87 |
| Q01484 | Ankyrin-2 | 52.62 | 2.75 |
| P03973 | Antileukoproteinase | 701.53 | 28.03 |
| P63010 | AP-2 complex subunit beta | 338.39 | 2.35 |
| P02647 | Apolipoprotein A-I | 612.31 | 39.70 |
| P02652 | Apolipoprotein A-II | 886.78 | 69.00 |
| Q5FYB0 | Arylsulfatase J | 389.18 | 10.35 |
| Q8IYB8 | ATP-dependent RNA helicase SUPV3L1_ mitochondrial | 235.17 | 6.23 |
| P04280 | Basic salivary proline-rich protein 1 | 3925.20 | 58.67 |
| P02812 | Basic salivary proline-rich protein 2 | 73554.97 | 69.47 |
| P61769 | Beta-2-microglobulin | 3725.17 | 48.74 |
| Q562R1 | Beta-actin-like protein 2 | 1532.83 | 13.30 |
| P13929 | Beta-enolase | 264.78 | 13.36 |
| Q96DR5 | BPI fold-containing family A member 2 | 4561.18 | 58.23 |
| Q8N4F0 | BPI fold-containing family B member 2 | 6508.75 | 30.79 |
| A0A087WXK1 | BRCA1-A complex subunit Abraxas (Fragment) | 332.77 | 16.93 |
| Q8N4G4 | CA6 protein | 419.28 | 4.47 |
| O75638 | Cancer/testis antigen 2 | 716.39 | 19.05 |
| P23280 | Carbonic anhydrase 6 | 15792.21 | 62.01 |
| P00450 | Ceruloplasmin | 71.04 | 8.45 |
| E9PM92 | Chromosome 11 open reading frame 58 | 258.69 | 15.29 |
| P01024 | Complement C3 | 833.42 | 21.17 |
| P51160 | Cone cGMP-specific 3'_5'-cyclic phosphodiesterase subunit alpha' | 232.14 | 11.07 |
| H3BRY3 | Coronin | 502.10 | 22.11 |
| P31146 | Coronin-1A | 502.10 | 24.95 |
| Q92772 | Cyclin-dependent kinase-like 2 | 457.97 | 11.97 |
| P04080 | Cystatin-B | 2288.27 | 45.92 |
| P01034 | Cystatin-C | 3131.85 | 51.37 |
| P28325 | Cystatin-D | 3348.32 | 61.97 |
| P01036 | Cystatin-S | 34860.66 | 73.76 |
| P09228 | Cystatin-SA | 24277.69 | 67.38 |
| P01037 | Cystatin-SN | 23133.23 | 70.21 |
| P54108 | Cysteine-rich secretory protein 3 | 284.38 | 21.63 |
| P32320 | Cytidine deaminase | 1245.08 | 66.44 |
| Q9UGM3 | Deleted in malignant brain tumors 1 protein | 306.82 | 4.97 |
| Q13609 | Deoxyribonuclease gamma | 411.37 | 15.74 |
| A0A0A0MT68 | Deoxyribonuclease | 411.37 | 16.67 |
| P27487 | Dipeptidyl peptidase 4 | 73.31 | 4.83 |
| O60216 | Double-strand-break repair protein rad21 homolog | 322.95 | 19.02 |
| R4GN68 | Dual-specificity mitogen-activated protein kinase kinase 4 | 780.16 | 97.56 |
| V9HW75 | Epididymis secretory protein Li 109 | 954.67 | 25.48 |
| B1AK53 | Espin | 277.28 | 4.80 |
| Q01469 | Fatty acid-binding protein_ epidermal | 475.76 | 30.37 |
| Q8NCQ5 | F-box only protein 15 | 465.73 | 3.73 |
| P02679 | Fibrinogen gamma chain | 372.17 | 21.63 |
| Q08380 | Galectin-3-binding protein | 237.96 | 18.97 |
| P06744 | Glucose-6-phosphate isomerase | 222.14 | 22.04 |
| E7ETY7 | Glutathione peroxidase | 341.42 | 22.78 |
| P09211 | Glutathione S-transferase P | 519.29 | 25.71 |
| P04406 | Glyceraldehyde-3-phosphate dehydrogenase | 407.39 | 11.64 |
| Q8IWJ2 | GRIP and coiled-coil domain-containing protein 2 | 718.24 | 4.81 |
| P00738 | Haptoglobin | 960.32 | 41.87 |
| G3V1N2 | HCG1745306_ isoform CRA_a | 11936.33 | 57.27 |
| P69905 | Hemoglobin subunit alpha | 13598.42 | 54.93 |
| P68871 | Hemoglobin subunit beta | 18402.54 | 89.80 |
| P02042 | Hemoglobin subunit delta | 5838.89 | 63.95 |
| P02100 | Hemoglobin subunit epsilon | 3895.00 | 6.80 |
| P69891 | Hemoglobin subunit gamma-1 | 3895.00 | 6.80 |
| P69892 | Hemoglobin subunit gamma-2 | 3895.00 | 6.80 |
| P15515 | Histatin-1 | 16204.54 | 36.84 |
| P15516 | Histatin-3 | 2631.50 | 13.73 |
| Q16695 | Histone H3.1t | 524.06 | 23.53 |
| Q05469 | Hormone-sensitive lipase | 43.68 | 5.30 |
| Q4G0P3 | Hydrocephalus-inducing protein homolog | 15.21 | 1.93 |
| A0A0G2JMB2 | Ig alpha-2 chain C region (Fragment) | 43004.29 | 79.12 |
| A0A0A0MS07 | Ig gamma-1 chain C region (Fragment) | 2528.80 | 42.37 |
| A0A087WYJ9 | Ig mu chain C region | 4012.85 | 48.67 |
| P04220 | Ig mu heavy chain disease protein | 3190.64 | 37.85 |
| P01876 | Immunoglobulin heavy constant alpha 1 | 38140.46 | 73.65 |
| P01877 | Immunoglobulin heavy constant alpha 2 | 32255.84 | 65.29 |
| P01857 | Immunoglobulin heavy constant gamma 1 | 4336.06 | 47.88 |
| P01859 | Immunoglobulin heavy constant gamma 2 | 1181.17 | 37.42 |
| P01860 | Immunoglobulin heavy constant gamma 3 | 1276.14 | 14.59 |
| P01861 | Immunoglobulin heavy constant gamma 4 | 1489.84 | 38.23 |
| P01871 | Immunoglobulin heavy constant mu | 4017.99 | 50.33 |
| A0A075B7F0 | Immunoglobulin heavy variable 3/OR16-10 (non-functional) (Fragment) | 299.80 | 9.48 |
| A0A075B7B8 | Immunoglobulin heavy variable 3/OR16-12 (non-functional) (Fragment) | 242.49 | 9.40 |
| A0A075B7E8 | Immunoglobulin heavy variable 3/OR16-13 (non-functional) (Fragment) | 242.49 | 9.40 |
| S4R460 | Immunoglobulin heavy variable 3/OR16-9 (non-functional) | 5489.71 | 31.25 |
| P01762 | Immunoglobulin heavy variable 3-11 | 299.80 | 9.40 |
| P01766 | Immunoglobulin heavy variable 3-13 | 299.80 | 9.48 |
| A0A0C4DH32 | Immunoglobulin heavy variable 3-20 (Fragment) | 299.80 | 9.40 |
| A0A0B4J1V1 | Immunoglobulin heavy variable 3-21 | 299.80 | 9.40 |
| P01764 | Immunoglobulin heavy variable 3-23 | 242.49 | 12.82 |
| P01768 | Immunoglobulin heavy variable 3-30 | 242.49 | 31.62 |
| P01772 | Immunoglobulin heavy variable 3-33 | 242.49 | 31.62 |
| A0A0B4J1X8 | Immunoglobulin heavy variable 3-43 | 299.80 | 9.32 |
| P01763 | Immunoglobulin heavy variable 3-48 | 299.80 | 9.40 |
| P01767 | Immunoglobulin heavy variable 3-53 | 242.49 | 12.93 |
| A0A0C4DH42 | Immunoglobulin heavy variable 3-66 | 242.49 | 12.93 |
| P01780 | Immunoglobulin heavy variable 3-7 | 299.80 | 9.40 |
| A0A0B4J1X5 | Immunoglobulin heavy variable 3-74 | 242.49 | 9.40 |
| P01782 | Immunoglobulin heavy variable 3-9 | 299.80 | 9.32 |
| P01591 | Immunoglobulin J chain | 20006.96 | 49.06 |
| P01834 | Immunoglobulin kappa constant | 28856.88 | 82.24 |
| A0A0C4DH90 | Immunoglobulin kappa variable 3/OR2-268 (non-functional) (Fragment) | 362.90 | 7.76 |
| P04433 | Immunoglobulin kappa variable 3-11 | 1198.54 | 26.09 |
| P01624 | Immunoglobulin kappa variable 3-15 | 362.90 | 7.83 |
| A0A075B6H7 | Immunoglobulin kappa variable 3-7 (non-functional) (Fragment) | 362.90 | 7.76 |
| A0A0A0MRZ8 | Immunoglobulin kappa variable 3D-11 | 1198.54 | 26.09 |
| A0A0C4DH55 | Immunoglobulin kappa variable 3D-7 | 362.90 | 7.56 |
| P06312 | Immunoglobulin kappa variable 4-1 | 250.98 | 19.83 |
| P0CG04 | Immunoglobulin lambda constant 1 | 40610.55 | 77.36 |
| P0DOY2 | Immunoglobulin lambda constant 2 | 44714.51 | 93.40 |
| P0DOY3 | Immunoglobulin lambda constant 3 | 44714.51 | 93.40 |
| P0CF74 | Immunoglobulin lambda constant 6 | 23147.62 | 50.94 |
| A0M8Q6 | Immunoglobulin lambda constant 7 | 19435.36 | 36.79 |
| P01715 | Immunoglobulin lambda variable 3-1 | 344.58 | 38.26 |
| B9A064 | Immunoglobulin lambda-like polypeptide 5 | 40610.55 | 38.32 |
| Q9BQU0 | Inhibitory NK receptor | 242.62 | 11.21 |
| Q9NVH2 | Integrator complex subunit 7 | 267.39 | 4.26 |
| Q01638 | Interleukin-1 receptor-like 1 | 304.24 | 7.01 |
| H0YNL8 | Iron-responsive element-binding protein 2 | 377.91 | 29.09 |
| A0A0G2JPA6 | Killer cell immunoglobulin-like receptor 3DL2 | 242.62 | 11.64 |
| P22079 | Lactoperoxidase | 2259.91 | 35.11 |
| P02788 | Lactotransferrin | 862.74 | 28.59 |
| A6NMS7 | Leucine-rich repeat-containing protein 37A | 263.12 | 1.71 |
| A6NM11 | Leucine-rich repeat-containing protein 37A2 | 252.18 | 1.71 |
| O60309 | Leucine-rich repeat-containing protein 37A3 | 276.06 | 4.53 |
| P31025 | Lipocalin-1 | 14925.97 | 53.98 |
| Q86W92 | Liprin-beta-1 | 292.75 | 10.29 |
| P00338 | L-lactate dehydrogenase A chain | 196.57 | 21.69 |
| Q9BY66 | Lysine-specific demethylase 5D | 307.10 | 8.58 |
| P61626 | Lysozyme C | 15283.53 | 66.89 |
| P14174 | Macrophage migration inhibitory factor | 616.56 | 47.83 |
| C9JF79 | Malate dehydrogenase (Fragment) | 263.72 | 11.71 |
| P40925 | Malate dehydrogenase_ cytoplasmic | 653.55 | 11.38 |
| Q5HYA8 | Meckelin | 241.84 | 1.61 |
| Q9Y4B5 | Microtubule cross-linking factor 1 | 26.23 | 1.52 |
| Q8TAX7 | Mucin-7 | 13700.40 | 9.28 |
| U3KPS2 | Myeloblastin | 554.69 | 17.67 |
| P24158 | Myeloblastin | 631.43 | 28.52 |
| Q9NYA4 | Myotubularin-related protein 4 | 315.44 | 7.11 |
| P59665 | Neutrophil defensin 1 | 1789.52 | 25.53 |
| P59666 | Neutrophil defensin 3 | 1789.52 | 25.53 |
| Q9BYH8 | NF-kappa-B inhibitor zeta | 371.15 | 4.32 |
| Q2L696 | Nucb2 splice variant | 663.95 | 25.13 |
| A0A087WSV8 | Nucleobindin 2_ isoform CRA_b | 954.67 | 25.48 |
| P80303 | Nucleobindin-2 | 954.67 | 25.48 |
| P04746 | Pancreatic alpha-amylase | 88276.59 | 55.97 |
| P42338 | Phosphatidylinositol 4_5-bisphosphate 3-kinase catalytic subunit beta isoform | 561.79 | 6.26 |
| A0A0A0MRF9 | Phosphoinositide phospholipase C | 313.90 | 4.55 |
| P13796 | Plastin-2 | 283.93 | 25.04 |
| Q86YL7 | Podoplanin | 866.94 | 34.57 |
| P11940 | Polyadenylate-binding protein 1 | 582.59 | 10.69 |
| E7ERJ7 | Polyadenylate-binding protein | 582.59 | 11.26 |
| Q8NDX5 | Polyhomeotic-like protein 3 | 348.42 | 3.05 |
| P01833 | Polymeric immunoglobulin receptor | 12791.93 | 57.98 |
| Q8TCS8 | Polyribonucleotide nucleotidyltransferase 1_ mitochondrial | 32.49 | 3.19 |
| Q6S8J3 | POTE ankyrin domain family member E | 4118.47 | 13.86 |
| A5A3E0 | POTE ankyrin domain family member F | 4040.70 | 11.72 |
| P0CG38 | POTE ankyrin domain family member I | 3413.22 | 4.74 |
| P0CG39 | POTE ankyrin domain family member J | 2796.68 | 3.85 |
| Q8TED1 | Probable glutathione peroxidase 8 | 341.42 | 17.22 |
| Q8IZM9 | Probable sodium-coupled neutral amino acid transporter 6 | 426.92 | 6.80 |
| K7EJ44 | Profilin | 470.78 | 37.50 |
| P07737 | Profilin-1 | 910.82 | 49.29 |
| P12273 | Prolactin-inducible protein | 30448.27 | 76.71 |
| A0A0A0MT31 | Proline-rich protein 4 | 23475.68 | 72.29 |
| P07602 | Prosaposin | 510.46 | 39.12 |
| Q5W0V3 | Protein FAM160B1 | 862.81 | 23.66 |
| Q6P5S2 | Protein LEG1 homolog | 6592.09 | 36.97 |
| Q9H7Z3 | Protein NRDE2 homolog | 45.79 | 1.20 |
| P05109 | Protein S100-A8 | 3184.30 | 23.66 |
| P06702 | Protein S100-A9 | 1737.55 | 77.19 |
| O14795 | Protein unc-13 homolog B | 59.05 | 1.19 |
| H3BQ60 | Puratrophin-1 (Fragment) | 266.79 | 50.00 |
| Q9BYX7 | Putative beta-actin-like protein 3 | 2063.16 | 10.67 |
| Q5VSP4 | Putative lipocalin 1-like protein 1 | 3097.31 | 11.11 |
| P52566 | Rho GDP-dissociation inhibitor 2 | 1026.87 | 30.35 |
| P35913 | Rod cGMP-specific 3'_5'-cyclic phosphodiesterase subunit beta | 374.14 | 8.08 |
| P02810 | Salivary acidic proline-rich phosphoprotein 1/2 | 4566.91 | 72.29 |
| P02787 | Serotransferrin | 4566.92 | 48.42 |
| P02768 | Serum albumin | 63281.61 | 75.04 |
| O00193 | Small acidic protein | 258.69 | 13.11 |
| P02808 | Statherin | 41653.6 | 48.39 |
| P02814 | Submaxillary gland androgen-regulated protein 3B | 20898.6 | 65.82 |
| Q9UH99 | SUN domain-containing protein 2 | 70.82 | 1.67 |
| A0A075B6V5 | T cell receptor alpha variable 36/delta variable 7 (Fragment) | 278.89 | 24.78 |
| Q7Z6L1 | Tectonin beta-propeller repeat-containing protein 1 | 384.23 | 7.12 |
| F2Z350 | Testis-expressed protein 29 | 447.37 | 32.14 |
| Q7Z4L5 | Tetratricopeptide repeat protein 21B | 78.57 | 4.56 |
| P20061 | Transcobalamin-1 | 378.51 | 22.86 |
| P29401 | Transketolase | 676.10 | 30.98 |
| Q6ZMR5 | Transmembrane protease serine 11A | 281.15 | 11.16 |
| P02766 | Transthyretin | 438.46 | 44.22 |
| P60174 | Triosephosphate isomerase | 651.56 | 36.36 |
| O43818 | U3 small nucleolar RNA-interacting protein 2 | 297.84 | 16.00 |
| A0A0J9YY99 | Uncharacterized protein (Fragment) | 242.49 | 12.82 |
| H7C2Y3 | Uncharacterized protein C2orf80 (Fragment) | 318.87 | 16.41 |
| H0Y8H3 | Uncharacterized protein C3orf67 (Fragment) | 590.54 | 74.68 |
| A0A087WZY1 | Uncharacterized protein | 22581.8 | 16.60 |
| A0A0G2JMZ2 | Uncharacterized protein | 252.18 | 1.71 |
| A0A0G2JRT3 | Uncharacterized protein | 252.18 | 1.77 |
| P02774 | Vitamin D-binding protein | 245.21 | 21.52 |
| Q14508 | WAP four-disulfide core domain protein 2 | 935.99 | 33.87 |
| Q9UDV6 | Zinc finger protein 212 | 424.39 | 16.97 |
| P25311 | Zinc-alpha-2-glycoprotein | 2292.60 | 31.54 |
| Q96DA0 | Zymogen granule protein 16 homolog B | 46355.09 | 58.17 |
Table 4. Proteins of the saliva identified in only in the individual analysis.
| Accession number | Protein name | score | Cover(%) |
|---|---|---|---|
| P31947 | 14-3-3 protein sigma | 297.17 | 24.60 |
| O00231 | 26S proteasome non-ATPase regulatory subunit 11 | 453.07 | 10.66 |
| P68032 | Actin_ alpha cardiac muscle 1 | 7799.84 | 26.53 |
| P68133 | Actin_ alpha skeletal muscle | 7799.84 | 26.53 |
| P62736 | Actin_ aortic smooth muscle | 7555.95 | 23.61 |
| P60709 | Actin_ cytoplasmic 1 | 17763.84 | 65.60 |
| P63261 | Actin_ cytoplasmic 2 | 17763.84 | 65.60 |
| P63267 | Actin_ gamma-enteric smooth muscle | 7555.95 | 23.67 |
| Q0VD77 | ADAMTS-like protein 5 | 410.00 | 32.06 |
| P00813 | Adenosine deaminase | 350.67 | 12.67 |
| O60503 | Adenylate cyclase type 9 | 471.53 | 5.69 |
| Q99996 | A-kinase anchor protein 9 | 34.16 | 3.58 |
| C9JKR2 | Albumin_ isoform CRA_k | 29220.48 | 74.82 |
| P01009 | Alpha-1-antitrypsin | 413.67 | 11.24 |
| P01023 | Alpha-2-macroglobulin | 445.71 | 15.33 |
| A8K2U0 | Alpha-2-macroglobulin-like protein 1 | 148.51 | 10.32 |
| P04745 | Alpha-amylase 1 | 97076.24 | 78.86 |
| P19961 | Alpha-amylase 2B | 77429.32 | 62.82 |
| P06733 | Alpha-enolase | 1439.59 | 49.08 |
| Q8N6M6 | Aminopeptidase O | 261.58 | 10.13 |
| Q01484 | Ankyrin-2 | 39.24 | 4.22 |
| P02652 | Apolipoprotein A-II | 941.64 | 47.00 |
| Q14562 | ATP-dependent RNA helicase DHX8 | 365.21 | 7.38 |
| Q8IYB8 | ATP-dependent RNA helicase SUPV3L1_ mitochondrial | 331.22 | 7.00 |
| P04280 | Basic salivary proline-rich protein 1 | 8867.97 | 44.39 |
| P02812 | Basic salivary proline-rich protein 2 | 54196.77 | 69.71 |
| I3L192 | Basigin (Fragment) | 185.70 | 16.88 |
| P61769 | Beta-2-microglobulin | 2754.07 | 54.62 |
| Q562R1 | Beta-actin-like protein 2 | 1943.05 | 10.90 |
| P13929 | Beta-enolase | 131.58 | 7.60 |
| O95342 | Bile salt export pump | 495.58 | 8.18 |
| Q96DR5 | BPI fold-containing family A member 2 | 6426.16 | 43.37 |
| Q8N4F0 | BPI fold-containing family B member 2 | 6613.00 | 37.99 |
| Q9NQY0 | Bridging integrator 3 | 398.03 | 11.46 |
| Q8N4G4 | CA6 protein | 294.75 | 4.47 |
| O75808 | Calpain-15 | 215.66 | 3.68 |
| P23280 | Carbonic anhydrase 6 | 9824.04 | 57.47 |
| Q0P665 | CCDC88C protein | 188.41 | 0.00 |
| Q8N163 | Cell cycle and apoptosis regulator protein 2 | 573.49 | 11.05 |
| O14647 | Chromodomain-helicase-DNA-binding protein 2 | 250.16 | 2.84 |
| H0Y7A8 | Chromosome 9 open reading frame 3 (Fragment) | 236.18 | 19.31 |
| P35606 | Coatomer subunit beta' | 189.71 | 2.21 |
| A2ABG0 | Complement C2 (Fragment) | 409.38 | 20.25 |
| P01024 | Complement C3 | 526.68 | 24.53 |
| Q53SF7 | Cordon-bleu protein-like 1 | 168.78 | 4.32 |
| P04080 | Cystatin-B | 1041.42 | 70.41 |
| P01034 | Cystatin-C | 3437.76 | 51.37 |
| P28325 | Cystatin-D | 2141.16 | 75.35 |
| P01036 | Cystatin-S | 28189.63 | 76.60 |
| P09228 | Cystatin-SA | 13641.19 | 67.38 |
| P01037 | Cystatin-SN | 28293.31 | 70.21 |
| P54108 | Cysteine-rich secretory protein 3 | 373.11 | 34.29 |
| Q8NF50 | Dedicator of cytokinesis protein 8 | 351.74 | 5.72 |
| Q9UGM3 | Deleted in malignant brain tumors 1 protein | 285.95 | 7.05 |
| Q5TBH6 | Dihydroxyacetone phosphate acyltransferase (Fragment) | 182.78 | 23.42 |
| P28340 | DNA polymerase delta catalytic subunit | 269.07 | 5.15 |
| M0R2B7 | DNA polymerase | 269.07 | 5.03 |
| Q5T4S7 | E3 ubiquitin-protein ligase UBR4 | 22.83 | 2.70 |
| Q92838 | Ectodysplasin-A | 258.64 | 15.86 |
| Q8N3D4 | EH domain-binding protein 1-like protein 1 | 260.45 | 4.33 |
| Q6P179 | Endoplasmic reticulum aminopeptidase 2 | 522.88 | 7.92 |
| Q7L775 | EPM2A-interacting protein 1 | 277.97 | 2.80 |
| Q9H501 | ESF1 homolog | 205.30 | 12.22 |
| A0A1B0GUN9 | Espin | 59.79 | 6.02 |
| Q8IXL6 | Extracellular serine/threonine protein kinase FAM20C | 322.36 | 5.48 |
| Q01469 | Fatty acid-binding protein_ epidermal | 444.20 | 32.59 |
| Q9BZK7 | F-box-like/WD repeat-containing protein TBL1XR1 | 376.57 | 15.18 |
| P02675 | Fibrinogen beta chain | 187.44 | 13.03 |
| P15328 | Folate receptor alpha | 400.38 | 35.80 |
| Q8NHY3 | GAS2-like protein 2 | 287.31 | 6.14 |
| P06396 | Gelsolin | 427.99 | 17.77 |
| O14893 | Gem-associated protein 2 | 443.14 | 31.07 |
| P53611 | Geranylgeranyl transferase type-2 subunit beta | 470.85 | 16.92 |
| P06744 | Glucose-6-phosphate isomerase | 787.26 | 28.49 |
| P04406 | Glyceraldehyde-3-phosphate dehydrogenase | 793.86 | 39.40 |
| O95427 | GPI ethanolamine phosphate transferase 1 | 233.92 | 7.73 |
| Q8IWJ2 | GRIP and coiled-coil domain-containing protein 2 | 22.31 | 1.25 |
| P00738 | Haptoglobin | 1233.11 | 55.42 |
| P00739 | Haptoglobin-related protein | 281.28 | 15.52 |
| G3V1N2 | HCG1745306_ isoform CRA_a | 15851.36 | 94.55 |
| E7BWR8 | HCG2043595_ isoform CRA_a | 252.74 | 7.76 |
| P69905 | Hemoglobin subunit alpha | 16443.62 | 83.80 |
| P68871 | Hemoglobin subunit beta | 22740.65 | 95.24 |
| P02042 | Hemoglobin subunit delta | 5150.58 | 39.46 |
| P02100 | Hemoglobin subunit epsilon | 2097.61 | 6.80 |
| P69891 | Hemoglobin subunit gamma-1 | 2097.61 | 6.80 |
| P69892 | Hemoglobin subunit gamma-2 | 2097.61 | 6.80 |
| P15515 | Histatin-1 | 5208.41 | 36.84 |
| P15516 | Histatin-3 | 4795.66 | 13.73 |
| E9PRF4 | Histone-lysine N-methyltransferase (Fragment) | 316.72 | 3.89 |
| Q15047 | Histone-lysine N-methyltransferase SETDB1 | 316.72 | 3.80 |
| P47902 | Homeobox protein CDX-1 | 196.38 | 26.04 |
| P31270 | Homeobox protein Hox-A11 | 264.91 | 14.38 |
| P09630 | Homeobox protein Hox-C6 | 93.47 | 4.68 |
| Q4G0P3 | Hydrocephalus-inducing protein homolog | 264.63 | 2.46 |
| A0A0G2JMB2 | Ig alpha-2 chain C region (Fragment) | 48303.27 | 79.12 |
| A0A0A0MS07 | Ig gamma-1 chain C region (Fragment) | 3209.86 | 45.76 |
| A0A087WYJ9 | Ig mu chain C region | 3019.36 | 54.87 |
| P04220 | Ig mu heavy chain disease protein | 2170.36 | 39.90 |
| P01876 | Immunoglobulin heavy constant alpha 1 | 40927.72 | 84.42 |
| P01877 | Immunoglobulin heavy constant alpha 2 | 28394.92 | 68.53 |
| P01857 | Immunoglobulin heavy constant gamma 1 | 5891.82 | 50.91 |
| P01859 | Immunoglobulin heavy constant gamma 2 | 1360.10 | 31.29 |
| P01860 | Immunoglobulin heavy constant gamma 3 | 1756.61 | 30.24 |
| P01861 | Immunoglobulin heavy constant gamma 4 | 1509.92 | 30.89 |
| P01871 | Immunoglobulin heavy constant mu | 3019.36 | 54.75 |
| A0A075B7D0 | Immunoglobulin heavy variable 1/OR15-1 (non-functional) (Fragment) | 252.28 | 10.26 |
| A0A075B7F0 | Immunoglobulin heavy variable 3/OR16-10 (non-functional) (Fragment) | 3426.81 | 13.79 |
| S4R460 | Immunoglobulin heavy variable 3/OR16-9 (non-functional) | 8502.51 | 36.46 |
| P01762 | Immunoglobulin heavy variable 3-11 | 3426.81 | 23.08 |
| P01766 | Immunoglobulin heavy variable 3-13 | 3426.81 | 13.79 |
| A0A0C4DH32 | Immunoglobulin heavy variable 3-20 (Fragment) | 3426.81 | 13.68 |
| A0A0B4J1V1 | Immunoglobulin heavy variable 3-21 | 3426.81 | 23.08 |
| A0A0B4J1X8 | Immunoglobulin heavy variable 3-43 | 3426.81 | 13.56 |
| P01763 | Immunoglobulin heavy variable 3-48 | 3426.81 | 23.08 |
| P01780 | Immunoglobulin heavy variable 3-7 | 3426.81 | 23.08 |
| P01782 | Immunoglobulin heavy variable 3-9 | 3426.81 | 13.56 |
| A0A0B4J1U7 | Immunoglobulin heavy variable 6-1 | 294.24 | 5.79 |
| P01591 | Immunoglobulin J chain | 21280.25 | 68.55 |
| P01834 | Immunoglobulin kappa constant | 37053.21 | 85.98 |
| P04433 | Immunoglobulin kappa variable 3-11 | 1303.48 | 26.09 |
| P01619 | Immunoglobulin kappa variable 3-20 | 868.06 | 7.76 |
| A0A0A0MRZ8 | Immunoglobulin kappa variable 3D-11 | 1303.48 | 26.09 |
| P06312 | Immunoglobulin kappa variable 4-1 | 423.92 | 19.83 |
| P0CG04 | Immunoglobulin lambda constant 1 | 33910.90 | 77.36 |
| P0DOY2 | Immunoglobulin lambda constant 2 | 40674.07 | 77.36 |
| P0DOY3 | Immunoglobulin lambda constant 3 | 40674.07 | 77.36 |
| P0CF74 | Immunoglobulin lambda constant 6 | 30147.40 | 50.94 |
| A0M8Q6 | Immunoglobulin lambda constant 7 | 22557.57 | 36.79 |
| B9A064 | Immunoglobulin lambda-like polypeptide 5 | 33910.9 | 38.32 |
| P06870 | Kallikrein-1 | 196.20 | 10.31 |
| P43626 | Killer cell immunoglobulin-like receptor 2DL1 | 252.74 | 7.76 |
| A0A0G2JNJ6 | Killer cell immunoglobulin-like receptor 2DS1 | 325.76 | 16.62 |
| Q9HAQ2 | Kinesin-like protein KIF9 | 158.59 | 4.43 |
| B4DZK5 | Kinesin-like protein | 133.37 | 10.51 |
| Q6H2H3 | KIR2DL1 | 252.74 | 7.76 |
| P22079 | Lactoperoxidase | 1577.03 | 41.43 |
| P02788 | Lactotransferrin | 1069.99 | 35.21 |
| Q6PKG0 | La-related protein 1 | 139.16 | 6.20 |
| P09960 | Leukotriene A-4 hydrolase | 225.55 | 19.31 |
| P31025 | Lipocalin-1 | 8361.36 | 51.14 |
| P00338 | L-lactate dehydrogenase A chain | 986.52 | 20.78 |
| Q9BYZ2 | L-lactate dehydrogenase A-like 6B | 323.86 | 8.66 |
| Q9BY66 | Lysine-specific demethylase 5D | 59.78 | 0.78 |
| P61626 | Lysozyme C | 9288.56 | 54.05 |
| P14174 | Macrophage migration inhibitory factor | 254.18 | 55.65 |
| P14780 | Matrix metalloproteinase-9 | 225.62 | 15.13 |
| Q96JG8 | Melanoma-associated antigen D4 | 150.96 | 6.07 |
| P01033 | Metalloproteinase inhibitor 1 | 445.25 | 29.95 |
| Q96GX9 | Methylthioribulose-1-phosphate dehydratase | 198.99 | 23.14 |
| O15021 | Microtubule-associated serine/threonine-protein kinase 4 | 168.04 | 3.43 |
| O43283 | Mitogen-activated protein kinase kinase kinase 13 | 533.35 | 8.70 |
| Q8TAX7 | Mucin-7 | 10429.01 | 15.65 |
| Q8NI22 | Multiple coagulation factor deficiency protein 2 | 260.43 | 23.97 |
| O75970 | Multiple PDZ domain protein | 43.13 | 2.32 |
| P24158 | Myeloblastin | 341.23 | 17.19 |
| P59665 | Neutrophil defensin 1 | 2353.04 | 15.96 |
| P59666 | Neutrophil defensin 3 | 2353.04 | 15.96 |
| P04746 | Pancreatic alpha-amylase | 64829.77 | 60.27 |
| Q08752 | Peptidyl-prolyl cis-trans isomerase D | 470.08 | 17.57 |
| P13796 | Plastin-2 | 531.41 | 28.87 |
| P01833 | Polymeric immunoglobulin receptor | 16305.42 | 45.42 |
| Q6S8J3 | POTE ankyrin domain family member E | 3659.07 | 9.49 |
| A5A3E0 | POTE ankyrin domain family member F | 3575.10 | 10.14 |
| P0CG38 | POTE ankyrin domain family member I | 2591.40 | 5.67 |
| P0CG39 | POTE ankyrin domain family member J | 1362.79 | 4.82 |
| P17844 | Probable ATP-dependent RNA helicase DDX5 | 220.36 | 4.89 |
| I3L3D5 | Profilin (Fragment) | 1209.81 | 10.91 |
| P07737 | Profilin-1 | 1209.81 | 20.71 |
| P12273 | Prolactin-inducible protein | 22984.41 | 89.04 |
| A0A0A0MT31 | Proline-rich protein 4 | 52615.69 | 72.29 |
| P07602 | Prosaposin | 316.92 | 22.52 |
| Q9P219 | Protein Daple | 206.07 | 0.69 |
| P49354 | Protein farnesyltransferase/geranylgeranyltransferase type-1 subunit alpha | 1184.15 | 17.41 |
| Q6P5S2 | Protein LEG1 homolog | 7928.19 | 40.00 |
| Q9H7Z3 | Protein NRDE2 homolog | 339.41 | 6.79 |
| Q8WYL5 | Protein phosphatase Slingshot homolog 1 | 286.92 | 2.38 |
| O43663 | Protein regulator of cytokinesis 1 | 83.55 | 7.42 |
| P05109 | Protein S100-A8 | 1391.46 | 31.18 |
| P06702 | Protein S100-A9 | 2043.00 | 78.07 |
| Q9NQW1 | Protein transport protein Sec31B | 442.02 | 7.63 |
| Q92954 | Proteoglycan 4 | 188.50 | 2.78 |
| Q96MK3 | Pseudokinase FAM20A | 287.95 | 8.50 |
| Q9BYX7 | Putative beta-actin-like protein 3 | 1353.87 | 29.07 |
| Q5VSP4 | Putative lipocalin 1-like protein 1 | 3095.80 | 11.11 |
| Q5JXB2 | Putative ubiquitin-conjugating enzyme E2 N-like | 341.70 | 32.03 |
| A4QN01 | Putative uncharacterized protein encoded by LINC01553 | 191.02 | 19.53 |
| Q15276 | Rab GTPase-binding effector protein 1 | 211.79 | 8.58 |
| Q9Y2J0 | Rabphilin-3A | 47.85 | 7.93 |
| Q14699 | Raftlin | 796.05 | 17.30 |
| G3XAJ6 | Raft-linking protein_ isoform CRA_c | 779.81 | 13.84 |
| P52565 | Rho GDP-dissociation inhibitor 1 | 251.72 | 19.61 |
| Q8IXT5 | RNA-binding protein 12B | 263.79 | 6.39 |
| K4DI92 | RWD domain containing 4A | 636.75 | 30.48 |
| Q6NW29 | RWD domain-containing protein 4 | 636.75 | 30.32 |
| P02810 | Salivary acidic proline-rich phosphoprotein 1/2 | 52615.69 | 72.29 |
| Q9BZL6 | Serine/threonine-protein kinase D2 | 403.28 | 9.68 |
| B4DTS2 | Serine/threonine-protein kinase | 401.26 | 9.57 |
| P02787 | Serotransferrin | 4390.41 | 39.26 |
| P02768 | Serum albumin | 64055.35 | 79.80 |
| P02808 | Statherin | 25654.54 | 48.39 |
| P02814 | Submaxillary gland androgen-regulated protein 3B | 50678.11 | 65.82 |
| P00441 | Superoxide dismutase [Cu-Zn] | 1005.47 | 45.45 |
| H0YN01 | Talin-2 | 197.30 | 34.55 |
| Q92609 | TBC1 domain family member 5 | 344.39 | 5.16 |
| Q7Z6L1 | Tectonin beta-propeller repeat-containing protein 1 | 62.51 | 2.49 |
| Q6N022 | Teneurin-4 | 64.41 | 4.15 |
| P10599 | Thioredoxin | 300.36 | 32.38 |
| Q96J01 | THO complex subunit 3 | 335.46 | 20.51 |
| Q5JTD0 | Tight junction-associated protein 1 | 432.54 | 3.95 |
| P37837 | Transaldolase | 676.70 | 23.74 |
| P20061 | Transcobalamin-1 | 670.49 | 33.26 |
| A6H8Y1 | Transcription factor TFIIIB component B'' homolog | 67.01 | 6.17 |
| P29401 | Transketolase | 1109.18 | 29.05 |
| Q9C0B7 | Transport and Golgi organization protein 6 homolog | 101.09 | 8.78 |
| P60174 | Triosephosphate isomerase | 582.07 | 15.73 |
| P07437 | Tubulin beta chain | 251.86 | 5.86 |
| Q13885 | Tubulin beta-2A chain | 268.91 | 5.84 |
| Q9BVA1 | Tubulin beta-2B chain | 251.86 | 5.84 |
| P04350 | Tubulin beta-4A chain | 242.62 | 5.86 |
| P68371 | Tubulin beta-4B chain | 242.62 | 5.84 |
| H3BLT7 | Tubulin monoglycylase TTLL3 (Fragment) | 205.55 | 1.15 |
| Q9NVE5 | Ubiquitin carboxyl-terminal hydrolase 40 | 49.55 | 6.15 |
| Q70EL2 | Ubiquitin carboxyl-terminal hydrolase 45 | 709.84 | 12.04 |
| D6RC01 | Ubiquitinyl hydrolase 1 | 685.20 | 10.14 |
| B4DSH7 | UDP-galactose translocator | 296.27 | 22.16 |
| H7C2Y3 | Uncharacterized protein C2orf80 (Fragment) | 203.05 | 50.78 |
| Q9H1L0 | Uncharacterized protein MIR1-1HG | 440.61 | 32.48 |
| A0A087WZY1 | Uncharacterized protein | 50162.86 | 16.60 |
| J3QRI8 | UPF0183 protein C16orf70 (Fragment) | 350.13 | 32.65 |
| Q13488 | V-type proton ATPase 116 kDa subunit a isoform 3 | 105.99 | 9.40 |
| Q14508 | WAP four-disulfide core domain protein 2 | 2122.26 | 33.87 |
| Q9NXC5 | WD repeat-containing protein mio | 208.07 | 1.94 |
| Q9BUG6 | Zinc finger and SCAN domain-containing protein 5A | 97.41 | 13.71 |
| Q8N8U3 | Zinc finger CCHC domain-containing protein 5 | 189.02 | 7.79 |
| Q9H0M4 | Zinc finger CW-type PWWP domain protein 1 | 242.57 | 7.10 |
| Q9NWS9 | Zinc finger protein 446 | 77.75 | 7.56 |
| P25311 | Zinc-alpha-2-glycoprotein | 1420.80 | 28.19 |
| Q96DA0 | Zymogen granule protein 16 homolog B | 32673.11 | 56.73 |
Discussion
This study aimed at standardizing a protocol for proteomic analysis of saliva that is sensitive, easy to perform and of low cost, to be used in future experiments involving quantitative shotgun proteomics. The first issue to be solved was related to the necessity of depletion of highly abundant proteins in saliva, such as albumin and IgG 8 , 14 that could mask and make difficult the identification of low abundance biomarkers. Krief and collaborators 7 (2011) evaluated whether depletion of salivary amylase, albumin and IgGs could improve the ability to visualize proteins in two-dimensional gel electrophoresis (2-DE) in oral fluids. They observed 36 new spots after depletion, and 58 spots showed more than twofold increase intensity after depletion 7 . Therefore, we hypothesized that this better identification profile could occur not only in two-dimensional gel electrophoresis (2-DE), but also in shotgun proteomics, when albumin and IgG were depleted. Thus, in the first test, we compared the use or not of the albumin and IgG depletion column after the extraction process of the salivary proteins. For this, we used a pool of ten saliva samples. When the column was used, only 35 proteins were identified (Table 1). This figure increased to 248 when the column was not used (Table 2). We believe this occurred because, when using the albumin and IgG depletion column, there was also depletion of other proteins, since using the column increases one more process in the methodology. We also believe that many proteins could bind to albumin and IgGs, thus being depleted together. Among the identified proteins, in both situations, are those typically found in saliva. By contrast, when the depletion column was used, classical salivary proteins such as 14-3-3 proteins, histatins, statherin, lactoperoxidase, lactotransferrin, lysozyme C, neutrophil defensins, protein S100A9, serotransferrin and some cystatin isoforms were not identified. Thus, contrary to what was observed in gel-based proteomics 7 , in shotgun proteomics the use of albumin and IgG depletion column impaired protein identification according to our workflow. Some studies, in spite of that, report advantages in using depletion columns when more than one workflow is employed 14 . However, this increases the time and cost of the analysis.
In the second test, we compared analysis of pooled samples (from ten individuals) versus individual analysis, without using the depletion column. In the individual analysis 239 proteins were identified (Table 4), while 212 proteins were identified in the pooled sample (Table 3). One-hundred and twenty three proteins were common to both groups (data not shown), and among them are most of the proteins typically found in saliva. The proteins exclusively found in the individual sample or in the pooled sample are not typically reported in saliva, which might be related to individual variation. The analysis of individual samples is important to allow confident comparison among the groups under study, especially in quantitative shotgun proteomics.
Generally, the methodologies used in proteomics are classified into two main categories: the bottom-up, which is also called shotgun proteomics, or top-down proteomics. Both methodologies have advantages and limitations, and their employment depends on the treatment given to the sample 9 . Shotgun proteomics is characterized by analyzing samples after proteolytic digestion in peptides, which is typically performed with trypsin 2 , 9 , while the top-down proteome of a sample involves analysis of intact proteins 9 . In shotgun proteomics, proteins from a complex mixture are digested, and the resulting peptides are analyzed by mass spectrometry. One of the advantages of this strategy is to investigate a large number of proteins regardless of their size. The limitations are related to incomplete coverage of the protein sequence, loss of post-translational modifications and degradation because of proteolytic digestion 4 , 9 . The top-down proteomics differs from the shotgun as it explores intact proteins by injecting the proteins into the mass spectrometer without performing digestion, minimizing any change in the sample and allowing a better characterization of post-translational modifications, especially those related to naturally occurring cleavages and alternative splicing 3 , avoiding interference problems based on peptides and allowing deducing the primary structure of the protein 4 , 9 . However, this technique is considered bounded by the collision energy required in protein fragmentation, which is insufficient for proteins greater than 50 KDa, and its application is restricted to the analysis of purified proteins 4 , 9 , 11 . In addition, top-down proteomics method requires the use of one or more forms of separation prior to mass spectrometry analysis 12 . Moreover, top-down platforms are intrinsically limited by the sample treatments required for use in mass spectrometry, involving the use of acids such as formic and trifluoroacetic acid 9 , 12 , 19 , which inevitably exclude proteins that are insoluble in acidic solutions. In addition, intact high molecular weight proteins and heterogeneous glycosylated proteins are not accessible in their naturally occurring form, even to the best level of mass spectrometry 2 .
Previous studies demonstrated that top-down platforms cannot achieve the same coverage of shotgun platforms for different reasons, such as: i) the intact protein must be soluble in the acid solution compatible with an ESI-MS analysis; (ii) the protein should not be heterogeneous (glycosylated isoforms), because in this case the intact protein mass cannot be deduced by the ESI spectrum; (iii) protein dimensions have to be limited, because MS-MS fragmentation spectra are too complex to be interpreted 3 , 15 . Nonetheless, the top-down strategy may reveal the richness of the isoform and the diversity of post-translational modifications, which in the shotgun proteomics strategy may result in the relevant loss of this molecular information 2 , 3 . Thus, shotgun proteomics may exhibit this deficiency in the human saliva proteome, in which many proteins such as basic PRPs and acids are not very susceptible to the proteolytic enzymes action and reveal very similar sequences. Therefore, many fragments cannot be related to a specific original protein. However, the shotgun platforms showed the best performance in terms of number of components detected, because the sensitivity of mass spectrometry is sufficient to reveal thousands of peptides in a single analysis. In this way, shotgun proteomics covers the largest variety of detectable components, regardless of their mass, due to the proteolytic digestion of large proteins almost always generates peptides that can disclose the presence of the protein in a complex mixture. Due to these reasons, the number of salivary components currently detectable by shotgun proteomics approaches is more than five times greater than that of components detected by any other platform 2 , 10 . Thus, in this study we employed shotgun proteomics.
Based on the results of the two tests, the protocol for salivary shotgun proteomic analysis was satisfactory, since it allowed the identification of many proteins, including those typically found in saliva. Moreover, it is easy to perform and cheaper than the methods previously described, since it does not require the use of depletion columns. Furthermore, it allows individual analysis of the samples, which is very important in quantitative proteomics. Thus, this protocol could be used in future studies involving shotgun proteomic analysis of saliva.
Acknowledgements
The authors thank FAPESP for the concession of a scholarship to the first author (Proc. 2017/05031-2).
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