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Salivary proteome and peptidome profiling in type 1 diabetes mellitus using a quantitative approach Armando Caseiro, Rita Ferreira, Ana Padrão, Cláudio Quintaneiro, Amélia Pereira, Rosário Marinheiro, Rui Vitorino, and Francisco Amado J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/pr3010343 • Publication Date (Web): 13 Feb 2013 Downloaded from http://pubs.acs.org on February 16, 2013
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Journal of Proteome Research
Salivary proteome and peptidome profiling in type 1 diabetes mellitus using a quantitative approach Armando Caseiro1,2, Rita Ferreira1, Ana Padrão1, Cláudio Quintaneiro3, Amélia Pereira3, Rosário Marinheiro3, Rui Vitorino1, Francisco Amado1,4 1
QOPNA, Mass spectrometry center, Department of Chemistry, University of Aveiro,
Portugal 2
College of Health Technology of Coimbra, Polytechnic Institute of Coimbra, Coimbra,
Portugal. 3
Figueira da Foz Hospital, Internal Medicine Service, Portugal
4
School of Health Sciences, University of Aveiro, Portugal
Correspondence, Rui Vitorino Address: Department of Chemistry, University of Aveiro, 3810-193, Aveiro, Portugal E-mail:
[email protected] Fax: +351234370084
Running title: Salivary proteomics and peptidomics in diabetes Keywords: Quantitation, LC-MS/MS, nephropathy, retinopathy, saliva
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Abstract In the present study we applied iTRAQ-based quantitative approach to explore the salivary proteome and peptidome profile in selected subjects with type 1 diabetes, with and without micro-vascular complications, aiming to identify disease-related markers. From a total of 434 distinct proteins, bactericidal/permeability-increasing protein-like 1 and pancreatic adenocarcinoma up-regulated factor were found in higher levels in the saliva of all patients while increased content of other proteins like alpha-2macroglobulin, defensin alpha 3 neutrophil-specific, leukocyte elastase inhibitor, matrix metalloproteinase-9, neutrophil elastase, plastin-2, protein S100-A8 and protein S100A9 were related with micro-vascular complications as retinopathy and nephropathy. Protein-protein interaction network analysis suggests the functional clusters defense, inflammation and response to wounding as the most significantly associated with type 1 diabetes pathogenesis. Peptidome data not only support a diabetes-related higher susceptibility of salivary proteins to proteolysis (mainly of aPRP, bPRP1 and bPRP2) but also evidenced an increased content of some specific protein fragments known to be related with bacterial attachment and the accumulation of phosphopeptides involved in tooth protection. In overall, the salivary protein and peptide profile highlights the importance of the innate immune system in the pathogenesis of type 1 diabetes mellitus and related complications. This study provides an integrated perspective of salivary proteome and peptidome that should be further explored in future studies targeting specific disease markers.
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1. Introduction
In recent years, saliva has attracted widespread interest as a diagnostic fluid.1-7 Salivary composition generally reflects the health status of an individual or disease susceptibility for oral and systemic pathologies. The advantages of saliva in comparison with other bodily fluids for diagnostic purposes are given by its accessibility, noninvasive and easy collection. The recent advancements in proteomic technologies hold special promise in the use of saliva to explore novel biomarkers and therapeutic targets.8,
9
Indeed, new
potential diagnostic salivary markers of oral and systemic diseases as dental and gingival pathology, salivary gland disease, Sjögren syndrome, diabetes, head and neck carcinoma, breast and gastric cancers, sclerosis and psychiatric and neurological diseases have been proposed based on proteomic approaches.3,
6-8, 10-12
Nevertheless,
little emphasis has been given to salivary proteome analysis in subjects with diabetes, namely in type 1 diabetes mellitus (DM)11, 13 and no study is known that quantitatively evaluated the saliva proteome and peptidome changes related with this pathological condition. The importance of quantitative proteomics has been increasingly recognized aiming to provide useful information for clinical applications once it screens nonphysiological levels of certain proteins and/or peptides that may reflect pathological conditions.3 In order to evaluate the effect of more than 12 years of type 1 DM and related complications on their salivary proteome and peptidome, we performed a iTRAQ analysis using pooled saliva samples collected from four groups of individuals (subjects with no diagnosed diabetes-related complications; with nephropathy and retinopathy; with retinopathy; and healthy individuals) to identify potential protein and peptide targets for disease diagnosis. The results obtained highlight the involvement of an
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inflammatory and immune system response in the pathogenesis of type 1 DM, with higher amounts of proteins like alpha-2-macroglobulin, MMP-9, S100A8 and S100A9 found in the saliva of patients with micro-vascular complications. Salivary peptidome data not only support a DM-related higher susceptibility of salivary proteins to proteolysis but also evidence an increased content of some specific protein fragments known to be related with bacterial attachment and the accumulation of phosphopeptides that seem to be involved in tooth protection against erosion, more frequent in subjects with diabetes.
2. Material and Methods
2.1. Participating subjects Subjects enrolled in the present study included 15 type 1 DM patients: 5 with retinopathy and nephropathy (T1D-R+N), 5 with retinopathy (T1D-R) and 5 without chronic complications (T1D) followed-up by the internal medicine service of Hospital Distrital da Figueira da Foz – Portugal. Five healthy volunteers (Ctrl) were also included in the study. All groups were matched by gender and age. Subjects with diabetes presented disease duration of a minimum of 15 years, with HbA1c levels higher than 7.7 %, significantly different from healthy individuals (T1D-R+N vs Ctrl group (p
