Identification of Sjögren's syndrome oral fluid biomarker candidates following high-abundance protein depletion.

OBJECTIVE: SS is an autoimmune exocrinopathy affecting ∼1 million patients in the USA that is diagnosed mostly in middle-aged women. Oral fluids (OFs) serving as the mirror of the body were suggested as an ideal non-invasive diagnostic tool. Previously we developed depletion techniques for OF high-abundance proteins to increase visualization of low-abundance proteins. Therefore the aim of this study was to examine the effect of depletion pretreatments on the identification potential of SS OF biomarker candidates. METHODS: Unstimulated OFs were collected from 18 female SS patients and 18 healthy age- and gender-matched controls. High-abundance proteins were depleted using affinity and immunodepletion methodologies followed by semi-quantitative two-dimensional gel electrophoresis and quantitative dimethylation liquid chromatography tandem mass spectrometry (LC-MS/MS). To initially validate the MS results, western blotting was performed. RESULTS: The use of depletion strategy before proteomics analysis increased identification ability by 3-fold. Overall, 79 biomarker candidates were identified. Proteins with the most pronounced fold changes were related to SS serum or tissue factors. In addition, bioinformatics analysis of proteins with a >3-fold increase in SS patients showed calcium-binding proteins, defence-response proteins, proteins involved in apoptotic regulation, stress-response proteins and cell motion-related proteins. Preliminary validation by western blotting of profilin and CA-I indicated similar expression profile trends to those identified by quantitative MS. CONCLUSION: The significance of OF novel depletion methodologies is clearly demonstrated for increased visibility of biomarker candidates as well as for unveiling possible mechanisms involved in this syndrome. This represents a major contribution to our ability to use OF as a future diagnostic fluid.

High-efficiency immunomagnetic isolation of solid tissue-originated integrin-expressing adult stem cells.

Isolation of highly pure specific cell types is crucial for successful adult stem cell-based therapy. As the number of such cells in adult tissue is low, an extremely efficient method is needed for their isolation. Here, we describe cell-separation methodologies based on magnetic-affinity cell sorting (MACS) MicroBeads with monoclonal antibodies against specific membrane proteins conjugated to superparamagnetic particles. Cells labeled with MACS MicroBeads are retained in a magnetic field within a MACS column placed in a MACS separator, allowing fast and efficient separation. Both positively labeled and non-labeled fractions can be used directly for downstream applications as the separated cell fractions remain viable with no functional impairment. As immunomagnetic separation depends on the interaction between a cell's membrane and the magnetically labeled antibody, separation of specific cells originating from solid tissues is more complex and demands a cell-dissociating pretreatment. In this paper, we detail the use of immunomagnetic separation for the purpose of regenerating damaged salivary gland (SG) function in animal and human models of irradiated head and neck cancer. Each year 500,000 new cases of head and neck cancer occur worldwide. Most of these patients lose SG function following irradiation therapy. SGs contain integrin α6ß1-expressing epithelial stem cells. We hypothesized that these cells can be isolated, multiplied in culture and auto-implanted into the irradiated SGs to regenerate damaged SG function.

Proteomic profiling of whole-saliva reveals correlation between Burning Mouth Syndrome and the neurotrophin signaling pathway.

Burning mouth syndrome (BMS) is characterized by a spontaneous and chronic sensation of burning in the oral mucosa, with no apparent signs. The underlying pathophysiological and neuropathic mechanisms remain unclear. Here, we attempt to elucidate some of these mechanisms using proteomic profiling and bioinformatic analyses of whole-saliva (WS) from BMS patients compared to WS from healthy individuals. Qualitative and quantitative proteomic profiling was performed using two dimensional gel electrophoresis (2-DE) and quantitative mass spectrometry (q-MS). In order to improve protein visibility, 21 high abundance proteins were depleted before proteomic profiling. Quantitative proteomic analysis revealed 100 BMS specific proteins and an additional 158 proteins up-regulated by more than threefold in those with BMS. Bioinformatic analyses of the altered protein expression profile of BMS group indicated high correlations to three cellular mechanisms including the neurotrophin signaling pathway. Based on this finding, we suggest that neurotrophin signaling pathway is involved in the pathophysiology of BMS by amplifying P75NTR activity, which in turn increases neural apoptosis thereby reducing sub-papillary nerve fiber density in the oral mucosa.

Identification of salivary protein biomarkers for orthodontically induced inflammatory root resorption.

PURPOSE: Orthodontically induced inflammatory root resorption (OIIRR) is one of the most prevalent and unavoidable consequence of orthodontic tooth movement. The aim of this study was to discover potential diagnostic protein biomarkers for detection of OIIRR in whole saliva (WS). MATERIAL AND METHODS: Unstimulated WS was collected from 72 subjects: 48 OIIRR patients and 24 untreated, generally healthy, age and gender matched controls. Radiographic assessment of periapical x-rays of four upper incisors taken before and 9 months after bonding was done. High-abundance proteins were depleted followed by two-dimensional-gel-electrophoresis and quantitative mass spectrometry (qMS). Finally, to initially validate qMS results, Western blotting was performed. RESULTS: qMS revealed differentially expressed proteins in the moderate-to-severe OIIRR group, which have never been found in WS before. Additionally, in the moderate-to-severe young OIIRR group, the pathogenetic mechanisms related to actin cytoskeleton regulation and Fc gamma R- mediated phagocytosis were detected, while in adults- to focal adhesion. Preliminary validation by Western blotting of fetuin-A and p21-ARC indicated expression profile trends similar to those identified by qMS. CONCLUSION: The significance of WS novel proteomic methodologies is clearly demonstrated for detecting new OIIRR biomarkers as well as for unveiling possible novel pathogenetic mechanisms in both young and adult patients.

Comparison of diverse affinity based high-abundance protein depletion strategies for improved bio-marker discovery in oral fluids.

Oral fluids (OF) have been suggested as a source of biomarkers for oral and systemic health, but as with other bio-fluids, the presence of high-abundance proteins interferes with the detection of lower-abundance biomarkers. Here, we compared the performance of four depletion treatments: triple depletion (TD) of amylases, albumins and immunoglobulin G; multiple depletion (MD) of amylases and a panel of 20 proteins, a combination of the two (EMD) and combinatorial peptide ligand library based depletion termed CPLL. TD, MD, EMD and CPLL removed 76%, 83%, 85% and 94% of total proteins, respectively, coupled with increased low abundance protein detection and narrowed dynamic range. 2-DE revealed that all depletion pretreatments successfully clarified areas hampered by high-abundance proteins; however, EMD and CPLL exposed the highest number of proteins. Quantitative MS of EMD samples relative to none treated samples indicated that most of downregulated proteins (>90%) were EMD target proteins. In conclusion, a multiple step EMD and CPLL depletion approaches bring about the highest number of protein detection ability and the best hampered-area clearance. As CPLL requires at least 10 fold more protein starting material, we suggest EMD pretreatment as a new detection tool in instances of low protein starting material.

Effect of irradiation on cell transcriptome and proteome of rat submandibular salivary glands.

Salivary glands (SGs) are irreversibly damaged by irradiation (IR) treatment in head and neck cancer patients. Here, we used an animal irradiation model to investigate and define the molecular mechanisms affecting SGs following IR, focusing on saliva proteome and global transcription profile of submandibular salivary gland (SSG) tissue.We show that saliva secretion was gradually reduced to 50% of its initial level 12 weeks post-IR. Saliva protein composition was further examined by proteomic analysis following mass spectrometry (MS) analysis that revealed proteins with reduced expression originating from SSGs and proteins with increased expression derived from the serum, both indicating salivary tissue damage. To examine alterations in mRNA expression levels microarray analysis was performed. We found significant alterations in 95 genes, including cell-cycle arrest genes, SG functional genes and a DNA repair gene.Tissue damage was seen by confocal immunofluorescence of α-amylase and c-Kit that showed an increase and decrease, respectively, in protein expression. This was coherent with real-time PCR results.This data indicates that IR damages the SSG cells' ability to produce and secrete saliva and proteins, and maintain the physiological barrier between serum and saliva. The damage does not heal due to cell-cycle arrest, which prevents tissue regeneration. Taken together, our results reveal a new insight into IR pathobiology.

An approach to remove alpha amylase for proteomic analysis of low abundance biomarkers in human saliva.

Proteomic characterization of human whole saliva for the identification of disease-specific biomarkers is guaranteed to be an easy-to-use and powerful diagnostic tool for defining the onset, progression and prognosis of human systemic diseases and, in particular, oral diseases. The high abundance of proteins, mainly alpha amylase, hampers the detection of low abundant proteins appearing in the disease state and therefore should be removed. In the present study a 2-DE was used to analyze human whole saliva following the removal of alpha amylase by affinity adsorption to potato starch. After alpha amylase removal whole saliva was analyzed by SDS-PAGE showing at least sixfold removal efficiency and by an alpha amylase activity assay showing 97% reduced activity. MS identification of the captured alpha amylase after elution demonstrated specific removal; 2-DE analysis showed the selective removal of alpha amylase and consequently increased gel resolution. MS identification of protein spots in the 60 kDa area revealed 15 proteins, which were masked before alpha amylase removal. In conclusion, treatment of human whole saliva with an alpha amylase removal device increases gel resolution and enables a higher protein sample for analysis.