RAPID COMMUNICATION Finding new posttranslational modifications in salivary proline-rich proteins Rui Vitorino 1 , Renato Alves 1 , Anto ´nio Barros 1 , Armando Caseiro 1 , Rita Ferreira 1 , Maria Calheiros Lobo 2 , Ana Bastos 3 , Jose ´ Duarte 4 , Davide Carvalho 5 , Lu ´cio Lara Santos 3,6 and Francisco L. Amado 1 1 QOPNA, Mass Spectrometry Center, Department of Chemistry, University of Aveiro, Aveiro, Portugal 2 CESPU, Dental School, Northern Health Institute, Porto, Portugal 3 IPO-Porto, Portuguese Institute of Oncology Francisco Gentil, Porto, Portugal 4 CIAFEL, Faculty of Sports Science, University of Porto, Porto, Portugal 5 Endocrinology Department, S. Joa ˜ o Hospital, University of Porto Medical School, Porto, Portugal 6 University Fernando Pessoa, Porto, Portugal Received: April 21, 2010 Revised: July 9, 2010 Accepted: August 3, 2010 Proline-rich proteins (PRPs) are the most complex family of salivary peptides with distinct isoforms and PTMs. Up to date, only the serine phosphorylation at positions 8, 17, and 22 have been experimentally observed on acidic PRP (aPRPs), and at position 8 on basic PRP1 and 2. The presence of a glucoronyl group at Ser17 was also noticed on aPRP. The main goal of this study was to identify new PTMs and distinct isoforms of salivary PRPs using LC-MALDI-TOF/TOF. Through the salivary peptidome characterization of 20 different subjects from Control, Diabetic, and Head and Neck Cancer groups, it was possible to identify the following species: (i) N-glycosylation sites: two in basic proline-rich protein 2 (bPRP2), one in bPRP3 and one in bPRP4; (ii) O-glycosylation sites: two in bPRP2 and one in aPRP; (iii) other terminal monosaccharide sites: six in bPRP1, two in bPRP2 and two in bPRP3; (iv) other modifications such as N-terminal pyro-Glu (two in bPRP1, six in bPRP2, eight in bPRP3 and nine in bPRP4); (v) phosphorylation in serine, three in bPRP1, one in bPRP2, one in bPRP3 and one in aPRP1; (vi) bPRP1 (allele S, allele M and variant CP5) and bPRP4 (allele M). In summary, salivary peptidome data analysis allowed the identification of 45 new PRP- modified residues, mainly due to glycosylation, phosphorylation and conversion of Gln to pyro-Glu. Moreover, comparing all subject groups, it was noticed a predominance of N-acetyl hexosamine modification on bPRPs in the Head and Neck Cancer patients. Keywords: Biomedicine / LC-MS/MS / MALDI-TOF/TOF / Peptidomics / PTM / Saliva Salivary secretions, similarly to other body fluids, contain several low-molecular-weight protein species with the unique distinction that peptide species with an m/z up to 20 kDa are responsible for about 40% of the total secreted proteins [1]. Salivary peptides are mostly present as protein families; those that are closely related structurally [2, 3] have been grouped since the 1970s into a few major classes, namely, histatins, statherin, cystatins and proline-rich proteins (PRPs). PRPs, which comprise 20–30% of all saliva protein content [4], are divided into two different groups: acidic proline-rich proteins (aPRPs) and basic proline-rich proteins (bPRPs). The bPRPs are typically divided into glycosylated PRPs and nonglycosylated species [5]. The aPRPs complex is expressed by the PRH1 and PRH2 genes, which express the forms PRP1, PRP2, PIFs and Dbs that are proteolytically cleaved at the RPPR motif located at Abbreviations: aPRP, acidic proline-rich protein; bPRP, basic proline-rich protein; FDR, false discovery rate; HexNac, N-acetyl hexosamine; HNC, head and neck cancer; PRP, proline-rich protein Correspondence: Professor Francisco L. Amado, Department of Chemistry, University of Aveiro, 3810-193, Aveiro, Portugal E-mail: famado@ua.pt Fax: 1351234370084 & 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com 3732 Proteomics 2010, 10, 3732–3742 DOI 10.1002/pmic.201000261