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Original Article • J. Appl. Oral Sci. 26 • 2018 • https://doi.org/10.1590/1678-7757-2017-0561   copy

Standardization of a protocol for shotgun proteomic analysis of saliva

Authorship SCIMAGO INSTITUTIONS RANKINGS

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.

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 diseases1717 - Wang X, Kaczor-Urbanowicz KE, Wong DT. Salivary biomarkers in cancer detection. Med Oncol. 2017;34(1):7.. Therefore, saliva contains more than 2000 proteins and peptides that are involved in an infinity of different biological functions in the oral cavity1717 - Wang X, Kaczor-Urbanowicz KE, Wong DT. Salivary biomarkers in cancer detection. Med Oncol. 2017;34(1):7.. Saliva still plays a large role in the formation of acquired pellicle, which begins only a few seconds after exposure of the enamel to saliva55 - Hannig M. Ultrastructural investigation of pellicle morphogenesis at two different intraoral sites during a 24-h period. Clin Oral Investig. 1999;3(2):88-95..

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 plasma1313 - Schweigel H, Wicht M, Schwendicke F. Salivary and pellicle proteome: a datamining analysis. Sci Rep. 2016;6:38882..

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 diseases2020 - Xiao H, Wong DT. Proteomic analysis of microvesicles in human saliva by gel electrophoresis with liquid chromatography-mass spectrometry. Anal Chim Acta. 2012;723:61-7..

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 procedures66 - Jasim H, Olausson P, Hedenberg-Magnusson B, Ernberg M, Ghafouri B. The proteomic profile of whole and glandular saliva in healthy pain-free subjects. Sci Rep. 2016;6:39073.. 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 abundance1717 - Wang X, Kaczor-Urbanowicz KE, Wong DT. Salivary biomarkers in cancer detection. Med Oncol. 2017;34(1):7.,1818 - Winck FV, Prado Ribeiro AC, Ramos Domingues R, Ling LY, Riaño-Pachón DM, Rivera C, et al. Insights into immune responses in oral cancer through proteomic analysis of saliva and salivary extracellular vesicles. Sci Rep. 2015;5:16305.. 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 procedure77 - Krief G, Deutsch O, Gariba S, Zaks B, Aframian DJ, Palmon A. Improved visualization of low abundance oral fluid proteins after triple depletion of alpha amylase, albumin and IgG. Oral Dis. 2011;17(1):45-52.,88 - Krief G, Deutsch O, Zaks B, Wong DT, Aframian DJ, Palmon A. Comparison of diverse affinity based high-abundance protein depletion strategies for improved bio-marker discovery in oral fluids. J Proteomics. 2012;75(13):4165-75.. 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 health11 - Camisasca DR, da Rós Goncalves L, Soares MR, Sandim V, Nogueira FC, Garcia CH, et al. A proteomic approach to compare saliva from individuals with and without oral leukoplakia. J Proteomics. 2017;151:43-52..

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 health66 - Jasim H, Olausson P, Hedenberg-Magnusson B, Ernberg M, Ghafouri B. The proteomic profile of whole and glandular saliva in healthy pain-free subjects. Sci Rep. 2016;6:39073.,1313 - Schweigel H, Wicht M, Schwendicke F. Salivary and pellicle proteome: a datamining analysis. Sci Rep. 2016;6:38882.,2020 - Xiao H, Wong DT. Proteomic analysis of microvesicles in human saliva by gel electrophoresis with liquid chromatography-mass spectrometry. Anal Chim Acta. 2012;723:61-7..

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 studies1818 - Winck FV, Prado Ribeiro AC, Ramos Domingues R, Ling LY, Riaño-Pachón DM, Rivera C, et al. Insights into immune responses in oral cancer through proteomic analysis of saliva and salivary extracellular vesicles. Sci Rep. 2015;5:16305.. 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 studies66 - Jasim H, Olausson P, Hedenberg-Magnusson B, Ernberg M, Ghafouri B. The proteomic profile of whole and glandular saliva in healthy pain-free subjects. Sci Rep. 2016;6:39073.,1818 - Winck FV, Prado Ribeiro AC, Ramos Domingues R, Ling LY, Riaño-Pachón DM, Rivera C, et al. Insights into immune responses in oral cancer through proteomic analysis of saliva and salivary extracellular vesicles. Sci Rep. 2015;5:16305..

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)1616 - Ventura TM, Cassiano LP, Souza e Silva CM, Taira EA, Leite AL, Rios D, et al. The proteomic profile of the acquired enamel pellicle according to its location in the dental arches. Arch Oral Biol. 2017;79:20-9.. 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).

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Table 1
Salivary proteins identified when the albumin and IgG depletion column was used

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Table 2
Salivary proteins identified when the albumin and IgG depletion column was not used

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).

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Table 3
Proteins of the saliva identified in the pool analysis

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Table 4
Proteins of the saliva identified in only in the individual analysis

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 IgG88 - Krief G, Deutsch O, Zaks B, Wong DT, Aframian DJ, Palmon A. Comparison of diverse affinity based high-abundance protein depletion strategies for improved bio-marker discovery in oral fluids. J Proteomics. 2012;75(13):4165-75.,1414 - Sivadasan P, Gupta MK, Sathe GJ, Balakrishnan L, Palit P, Gowda H, et al. Human salivary proteome - a resource of potential biomarkers for oral cancer. J Proteomics. 2015;127(Pt A):89-95. that could mask and make difficult the identification of low abundance biomarkers. Krief and collaborators77 - Krief G, Deutsch O, Gariba S, Zaks B, Aframian DJ, Palmon A. Improved visualization of low abundance oral fluid proteins after triple depletion of alpha amylase, albumin and IgG. Oral Dis. 2011;17(1):45-52. (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 depletion77 - Krief G, Deutsch O, Gariba S, Zaks B, Aframian DJ, Palmon A. Improved visualization of low abundance oral fluid proteins after triple depletion of alpha amylase, albumin and IgG. Oral Dis. 2011;17(1):45-52.. 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 proteomics77 - Krief G, Deutsch O, Gariba S, Zaks B, Aframian DJ, Palmon A. Improved visualization of low abundance oral fluid proteins after triple depletion of alpha amylase, albumin and IgG. Oral Dis. 2011;17(1):45-52., 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 employed1414 - Sivadasan P, Gupta MK, Sathe GJ, Balakrishnan L, Palit P, Gowda H, et al. Human salivary proteome - a resource of potential biomarkers for oral cancer. J Proteomics. 2015;127(Pt A):89-95.. 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 sample99 - Manconi B, Liori B, Cabras T, Iavarone F, Manni A, Messana I, et al. Top-down HPLC-ESI-MS proteomic analysis of saliva of edentulous subjects evidenced high levels of cystatin A, cystatin B and SPRR3. Arch Oral Biol. 2017;77:68-74.. Shotgun proteomics is characterized by analyzing samples after proteolytic digestion in peptides, which is typically performed with trypsin22 - Castagnola M, Cabras T, Iavarone F, Vincenzoni F, Vitali A, Pisano E, et al. Top-down platform for deciphering the human salivary proteome. J Matern Fetal Neonatal Med. 2012;25(Suppl 5):27-43.,99 - Manconi B, Liori B, Cabras T, Iavarone F, Manni A, Messana I, et al. Top-down HPLC-ESI-MS proteomic analysis of saliva of edentulous subjects evidenced high levels of cystatin A, cystatin B and SPRR3. Arch Oral Biol. 2017;77:68-74., while the top-down proteome of a sample involves analysis of intact proteins99 - Manconi B, Liori B, Cabras T, Iavarone F, Manni A, Messana I, et al. Top-down HPLC-ESI-MS proteomic analysis of saliva of edentulous subjects evidenced high levels of cystatin A, cystatin B and SPRR3. Arch Oral Biol. 2017;77:68-74.. 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 digestion44 - Catherman AD, Skinner OS, Kelleher NL. Top Down proteomics: facts and perspectives. Biochem Biophys Res Commun. 2014;445(4):683-93.,99 - Manconi B, Liori B, Cabras T, Iavarone F, Manni A, Messana I, et al. Top-down HPLC-ESI-MS proteomic analysis of saliva of edentulous subjects evidenced high levels of cystatin A, cystatin B and SPRR3. Arch Oral Biol. 2017;77:68-74.. 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 splicing33 - Castagnola M, Scarano E, Passali GC, Messana I, Cabras T, Iavarone F, et al. Salivary biomarkers and proteomics: future diagnostic and clinical utilities. Acta Otorhinolaryngol Ital. 2017;37(2):94-101., avoiding interference problems based on peptides and allowing deducing the primary structure of the protein44 - Catherman AD, Skinner OS, Kelleher NL. Top Down proteomics: facts and perspectives. Biochem Biophys Res Commun. 2014;445(4):683-93.,99 - Manconi B, Liori B, Cabras T, Iavarone F, Manni A, Messana I, et al. Top-down HPLC-ESI-MS proteomic analysis of saliva of edentulous subjects evidenced high levels of cystatin A, cystatin B and SPRR3. Arch Oral Biol. 2017;77:68-74.. 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 proteins44 - Catherman AD, Skinner OS, Kelleher NL. Top Down proteomics: facts and perspectives. Biochem Biophys Res Commun. 2014;445(4):683-93.,99 - Manconi B, Liori B, Cabras T, Iavarone F, Manni A, Messana I, et al. Top-down HPLC-ESI-MS proteomic analysis of saliva of edentulous subjects evidenced high levels of cystatin A, cystatin B and SPRR3. Arch Oral Biol. 2017;77:68-74.,1111 - Nesatyy VJ, Suter MJ. Analysis of environmental stress response on the proteome level. Mass Spectrom Rev. 2008;27(6):556-74.. In addition, top-down proteomics method requires the use of one or more forms of separation prior to mass spectrometry analysis1212 - Peng Y, Chen X, Sato T, Rankin SA, Tsuji RF, Ge Y. Purification and high-resolution top-down mass spectrometric characterization of human salivary alpha-amylase. Anal Chem. 2012;84(7):3339-46.. 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 acid99 - Manconi B, Liori B, Cabras T, Iavarone F, Manni A, Messana I, et al. Top-down HPLC-ESI-MS proteomic analysis of saliva of edentulous subjects evidenced high levels of cystatin A, cystatin B and SPRR3. Arch Oral Biol. 2017;77:68-74.,1212 - Peng Y, Chen X, Sato T, Rankin SA, Tsuji RF, Ge Y. Purification and high-resolution top-down mass spectrometric characterization of human salivary alpha-amylase. Anal Chem. 2012;84(7):3339-46.,1919 - Wu S, Brown JN, Tolić N, Meng D, Liu X, Zhang H, et al. Quantitative analysis of human salivary gland-derived intact proteome using top-down mass spectrometry. Proteomics. 2014;14(10):1211-22., 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 spectrometry22 - Castagnola M, Cabras T, Iavarone F, Vincenzoni F, Vitali A, Pisano E, et al. Top-down platform for deciphering the human salivary proteome. J Matern Fetal Neonatal Med. 2012;25(Suppl 5):27-43..

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 interpreted33 - Castagnola M, Scarano E, Passali GC, Messana I, Cabras T, Iavarone F, et al. Salivary biomarkers and proteomics: future diagnostic and clinical utilities. Acta Otorhinolaryngol Ital. 2017;37(2):94-101.,1515 - Tipton JD, Tran JC, Catherman AD, Ahlf DR, Durbin KR, Kelleher NL. Analysis of intact protein isoforms by mass spectrometry. J Biol Chem. 2011;286(29):25451-8.. 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 information22 - Castagnola M, Cabras T, Iavarone F, Vincenzoni F, Vitali A, Pisano E, et al. Top-down platform for deciphering the human salivary proteome. J Matern Fetal Neonatal Med. 2012;25(Suppl 5):27-43.,33 - Castagnola M, Scarano E, Passali GC, Messana I, Cabras T, Iavarone F, et al. Salivary biomarkers and proteomics: future diagnostic and clinical utilities. Acta Otorhinolaryngol Ital. 2017;37(2):94-101.. 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 platform22 - Castagnola M, Cabras T, Iavarone F, Vincenzoni F, Vitali A, Pisano E, et al. Top-down platform for deciphering the human salivary proteome. J Matern Fetal Neonatal Med. 2012;25(Suppl 5):27-43.,1010 - Messana I, Cabras T, Iavarone F, Vincenzoni F, Urbani A, Castagnola M. Unraveling the different proteomic platforms. J Sep Sci. 2013;36(1):128-39.. 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).

References

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    - Camisasca DR, da Rós Goncalves L, Soares MR, Sandim V, Nogueira FC, Garcia CH, et al. A proteomic approach to compare saliva from individuals with and without oral leukoplakia. J Proteomics. 2017;151:43-52.
  • 2
    - Castagnola M, Cabras T, Iavarone F, Vincenzoni F, Vitali A, Pisano E, et al. Top-down platform for deciphering the human salivary proteome. J Matern Fetal Neonatal Med. 2012;25(Suppl 5):27-43.
  • 3
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    - Manconi B, Liori B, Cabras T, Iavarone F, Manni A, Messana I, et al. Top-down HPLC-ESI-MS proteomic analysis of saliva of edentulous subjects evidenced high levels of cystatin A, cystatin B and SPRR3. Arch Oral Biol. 2017;77:68-74.
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Publication Dates

  • Publication in this collection
    11 June 2018
  • Date of issue
    2018

History

  • Received
    14 Nov 2017
  • Reviewed
    22 Dec 2017
  • Accepted
    12 Jan 2018
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