Two-Dimensional Liquid Chromatography Study of the Human Whole Saliva Proteome

Wilmarth, Phillip A.; Riviere, Michael A.; Rustvold, D. Leif; Lauten, Jeffrey D.; Madden, Theresa E.; David, Larry L.

doi:10.1021/pr049911o

Cited by 136 publications

(134 citation statements)

References 42 publications

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“…Our results also show that only four saliva proteins were common in all specimens by the three methods. A previous saliva study using a two-dimensional (2D)-LC technique reported 102 proteins in their sample (28). The same method in our study yielded 118 saliva proteins.…”

Section: Methods 1 Methods 2 Methods 3 ---------------------------------supporting

confidence: 67%

“…The majority of these proteins were also found in normal salivas processed by the method except the ·-1-B-glycoprotein and complement factor B. None of these proteins have previously been reported in other studies of normal saliva (24)(25)(26)28,29). However, our Western blotting detected low levels of complement factor B in saliva of 2 of the 5 normal salivas.…”

Section: Methods 1 Methods 2 Methods 3 ---------------------------------supporting

confidence: 60%

“…The increasing interest in proteomic profiling of saliva has led to several attempts to characterize its proteins (24)(25)(26)(27)(28)(29). In these studies, only a limited number of normal specimens with varied collection and processing methods were analyzed.…”

Section: Introductionmentioning

confidence: 99%

“…However, major obstacles remain to complicate and impede the clinical implementation of this technology in human materials. These include sample-related factors such as quality, timing, composition, sample collection and processing, technical limitations and the complexities and the dynamic variations of human proteins (22,23).The increasing interest in proteomic profiling of saliva has led to several attempts to characterize its proteins (24)(25)(26)(27)(28)(29). In these studies, only a limited number of normal specimens with varied collection and processing methods were analyzed.…”

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confidence: 99%

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Pre-analytic saliva processing affect proteomic results and biomarker screening of head and neck squamous carcinoma

et al. 2007

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Abstract. The objective of this study was to assess the effect of pre-analytical processing on proteomic analysis of saliva and to identify salivary biomarkers for potential clinical applications. Saliva samples from five healthy individuals and three head and neck squamous carcinoma (HNSC) patients were initially depleted of major protein constituents. Saliva from healthy subjects was divided and processed by three different methods prior to liquid chromatography and tandem mass spectrometry technique (LC-MS/MS) analysis. The results showed marked differences amongst the methods. The SDS-PAGE separation and in-gel digestion method yielded the highest number of proteins that included the majority of those identified by the other two methods. The in geldigestion method was used in the LC-MS/MS analysis of saliva from three HNSC patients and the results were compared with those from healthy subjects. Our analysis identified two proteins, ·-1-B-glycoprotein and complement factor B proteins, to be present in patients but not in normal specimens. Paradoxically, cystatin S, parotid secretory factor, and poly-4-hydrolase ß-subunit proteins were detected in most normal salivas but not in patient specimens. Subsequent analysis of complement factor B by Western blotting showed strong immunoreactive bands of complement factor B in HNSC patients' and negative or weakly positive in normal saliva samples. We conclude that: 1) initial saliva processing affects protein analysis, 2) in-gel digestion followed by LC-MS/MS detects the most saliva proteins, 3) certain proteins are differentially found in patient and normal salivas and 4) a small set of proteins can be targeted for future validation for clinical investigation. IntroductionSaliva is a readily accessible secretion that plays an important role in the maintenance of oral homeostasis, tooth integrity, initiation of digestion, and antimicrobial defense (1-3). Saliva is protein rich and may constitute a valuable source of diagnostic and therapeutic markers in head and neck squamous cell carcinoma (HNSC) patients (4-6). The major components of saliva are mucins, proline-rich glycoproteins, amylase, lipase and a variety of antimicrobial proteins and peptides that include agglutinin, cystatins, lisozyme, lactoferrin, immunoglobulins, histatins and defensins (7-17). Numerous less common proteins derived from either active salivary secretion or through plasma filtration have also been identified (18-21). Analysis of these proteins may provide important information in health and diseased states.It has recently been acknowledged that proteomic analysis reflects the net sum of the metabolic and functional states of cells and secretions from tissues (22). Advances in both technological and bioinformatic fields have led to a plethora of efforts to identify diagnostic, prognostic and therapeutic targets using human secretions and body fluids (4). However, major obstacles remain to complicate and impede the clinical implementation of this technology in human materials. These include sa...

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Section: Methods 1 Methods 2 Methods 3 ---------------------------------supporting

confidence: 67%

Section: Methods 1 Methods 2 Methods 3 ---------------------------------supporting

confidence: 60%

Section: Introductionmentioning

confidence: 99%

mentioning

confidence: 99%

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Pre-analytic saliva processing affect proteomic results and biomarker screening of head and neck squamous carcinoma

et al. 2007

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“…Although it has the greatest protein resolving power of any available separation technique, 2-D GE has some limitations such as difficulties encountered in observing large or small molecular weight proteins, highly acidic or basic proteins, and hydrophobic proteins. Due to these limitations and the recent advances in several types of MS, 2-D LC coupled to MS methods are being increasingly adopted for separation of complex protein and protein digest samples [9][10][11][12][13][14]. The unique feature of the 2-D methods based on LC compared to 2-D GE is the relative ease with which they can be interfaced to MS analysis, especially when capillary LC columns are used.…”

Section: Introductionmentioning

confidence: 99%

Fractionation and separation of human salivary proteins by pH‐gradient ion exchange and reversed phase chromatography coupled to mass spectrometry

Pepaj¹,

Holm

Fleckenstein

et al. 2006

J of Separation Science

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Fractionation and separation of human salivary proteins by pH-gradient ion exchange and reversed phase chromatography coupled to mass spectrometryIn the present work, a 2-D capillary liquid chromatography method for fractionation and separation of human salivary proteins is demonstrated. Fractionation of proteins according to their pI values was performed in the 1-D employing a strong anion exchange (SAX) column subjected to a wide-range descending pH gradient. Polystyrene-divinylbenzene (PS-DVB) RP columns were used for focusing and subsequent separation of the proteins in the 2-D. The SAX column was presaturated with a high pH buffer (A) consisting of 10 mM amine buffering species, pH 9.0, and elution was performed with a low pH elution buffer (B) having the same buffer composition and concentration as buffer A, but pH 3.5. Isoelectric point fractions eluting from the 1-D column were trapped on PS-DVB trap columns prior to back-flushed elution onto the PS-DVB analytical column for separation of the proteins. The 1-D fraction eluting at pH 9.0-8.7 was chosen for further analysis. After separation on the RP analytical column, nine RP protein fractions were collected and tryptic digested for subsequent analyses by MALDI TOF MS and column switching capillary LC coupled to ESI TOF MS and ESI QTOF MS. Eight proteins and two peptides were identified in the pH 9.0-8.7 fraction using peptide mass fingerprinting and uninterpreted MS/MS data.

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Saliva in Health and Disease, Chemical Biology of

Fábían

Fejérdy²,

Csermely

2008

Wiley Encyclopedia of Chemical Biology

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Saliva is a bodily fluid secreted by three pairs of major salivary glands (parotid submandibular and sublingual) and by many of minor salivary glands. Saliva is supplemented with several constituents that originate from blood serum, from intact or destroyed mucosal and immune cells, and from intact or destroyed oral microorganisms that result in a complex mixture of a variety of molecules. Saliva plays an important role in acquired pellicle formation on tooth surfaces, crystal growth homeostasis, bacterial adhesion, plaque formation, and—because of its lubricating effect—in maintaining mucosal integrity of the oral and upper gastrointestinal mucosal surfaces. It also plays an important role in physico‐chemical defense, antimicrobial defense, and wound healing. Many saliva constituents including proteins, carbohydrates, lipids, and ions interact under fine regulation to fulfill these important tasks. Local and systemic disorders may disturb and interrupt these complex balanced functions, which can lead to mucosal and tooth damages. In other cases, systemic disorders induce salivary changes without any significant local effects. Many such changes are of high diagnostic interest because they can be rather specific to the causing conditions and can be used for screening and early diagnosis of several local and systemic disorders.

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Two-Dimensional Liquid Chromatography Study of the Human Whole Saliva Proteome

Cited by 136 publications

References 42 publications

Pre-analytic saliva processing affect proteomic results and biomarker screening of head and neck squamous carcinoma

Pre-analytic saliva processing affect proteomic results and biomarker screening of head and neck squamous carcinoma

Fractionation and separation of human salivary proteins by pH‐gradient ion exchange and reversed phase chromatography coupled to mass spectrometry

Saliva in Health and Disease, Chemical Biology of

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