Protease profiling of different biofluids in type 1 diabetes mellitus Armando Caseiro a, b , Rita Ferreira a , Cláudio Quintaneiro c , Amélia Pereira c , Rosário Marinheiro c , Rui Vitorino a, ⁎, Francisco Amado a, d a QOPNA, Mass Spectrometry Center, Department of Chemistry, University of Aveiro, Aveiro, Portugal b College of Health Technology of Coimbra, Polytechnic Institute of Coimbra, Coimbra, Portugal c Figueira da Foz Hospital, Internal Medicine Service, Figueira da Foz, Portugal d School of Health Sciences, University of Aveiro, Portugal abstract article info Article history: Received 17 April 2012 received in revised form 20 August 2012 accepted 26 August 2012 Available online 10 September 2012 Keywords: Zymography-LC-MS/MS Nephropathy Retinopathy Saliva Urine Objectives: We aimed to disclose the proteolytic events underlying type 1 diabetes and related compli- cation through protease profiling in the bodily fluids serum, urine and saliva. Design and methods: Zymography followed by LC-MS/MS was performed for protease identification and quantitative comparison of proteolytic activity between healthy, type 1 diabetic patients with no complications and with retinopathy and nephropathy. Western blotting was also accomplished for MMP-9 and MMP-2 identifi- cation and expression analysis. Results: Only MMP-2 and MMP-9 were observed in serum with significantly increased levels and activity ob- served in diabetic patients. In urine and saliva other proteases besides MMPs were identified by MS and presented disease-dependent activity variations. Among these are complex MMP-9/Neutrophil gelatinase-associated lipocalin, aminopeptidase N, azurocidin and kallikrein 1 with more activity noticed in type 1 diabetes patients with nephropathy and/or retinopathy. Conclusion: Our data highlight the usefulness of urine and saliva for the monitoring of type-1 diabetes-related proteolytic events, where aminopeptidase N, azurocidin and kallikrein 1 appear as promising screening targets for type 1 diabetes‐related complications. © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. Introduction Diabetes mellitus (DM) is a chronic debilitating disease affecting over 366 million of people worldwide [1,2]. Chronic hyperglycaemia is the critical factor for the development and progression of microvascular complications like nephropathy, retinopathy and peripheral neuropathy in diabetes [3–6]. Nevertheless, the molecular mechanisms influencing the severity of diabetic microvascular disease are not fully understood. Proteomic profiling of clinical species has been heralded as a powerful tool for the identification of altered biochemical pathways and bio- markers of disease states. More than 50 distinct proteins were already suggested as potential biomarkers in bodily fluids from DM patients. Al- terations in the levels of distinct apolipoproteins (e.g. A-1, J, C-1) were reported in the plasma and urine of type 1 and type 2 DM, whereas Zn-α-2-glycoprotein 1 and transthyretin were found up- and down- regulated, respectively, in the urine of type 2 DM [7–10]. However, little emphasis has been given to protease profiling in biofluids [11] and even less to its alterations in diabetes mellitus. Besides metalloproteases (MMPs) and kallikreins, no other proteases have been implicated in the pathophysiology of DM and related complications [12,13]. The increased serum levels of these zinc endopeptidases capable of degrading all the components of the extracellular matrix, particularly of MMP-9 and MMP-2, were already suggested as a marker of chronic kidney disease's risk [13] and of active retinopathy [14]. Recently, McKittrick et al. [15] suggested urinary MMP activities as clinically rele- vant biomarkers for predicting vascular remodeling in diabetic renal and vascular complications in type 1 DM patients. In type 2 DM, kallikrein 3 was found down-regulated in urine [10]. No other proteases have been related to DM pathogenesis though more than 500 human proteases are included in the degradome database and some have been suggested to have context-dependent disease roles [16]. In order to evaluate type 1 DM-related alterations on biofluids’ protease profile we performed a straightforward screening of the pro- teases present in saliva, serum and urine from diabetic patients with no complications diagnosed, with nephropathy and with retinopathy using zymography-LC-MS/MS. Material and methods Patients Subjects enrolled in the present study included 15 type 1 diabetic patients: 5 with retinopathy and nephropathy (group A), 5 with ret- inopathy (group B) and 5 without chronic complications (group C) Clinical Biochemistry 45 (2012) 1613–1619 ⁎ Corresponding author at: Department of Chemistry, University of Aveiro, 3810–193, Aveiro, Portugal. Fax: +351 234370084. E-mail address: rvitorino@ua.pt (R. Vitorino). 0009-9120/$ – see front matter © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.clinbiochem.2012.08.027 Contents lists available at SciVerse ScienceDirect Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem