Protein identification from salivary stones with MALDI-TOF mass spectrometry

1. Introduction: Sialolithiasis is a type of Lithiasis (a pathological formation of mineral concretions in the human body), where liths are formed in the salivary glands. It affects approximately 1% of the population, causing acute and chronic infections. The structure of the concrements can be homogenous or it is concentrically lamellar containing a central core region and a layered cortex. Sialoliths consist of both inorganic compounds (mainly calcium phosphate) and organic molecules. In the literature there are several contradictory theories about the composition and the spatial distribution of the organic constituents. Sialoliths were divided mechanically to central and peripheral part. The core and cortex region of salivary stones were grinded, extracted with urea, digested with trypsin and then analysed with MALDI-TOF (matrix assisted laser desorption ionisation - time of flight) instrument. Peptide mass finger prints were collected and compared with the results of in silico digestion. MS/MS measurements were completed from the intensive masses and the peptides were sequenced. The findings were compared with the results of the MASCOT Database search. Protein identification from salivary stones with MALDI-TOF mass spectrometry Katalin Böddi1, Ane S. Brochmann2, József Szalma2, Edina Lempel2, Zoltán Szabó1, Anikó Takátsy1 1) University of Pécs, Faculty of Medicine (Department of Biochemistry and Medical Chemistry, Pécs, Szigeti str.12) 2) University of Pécs (Department of Oral and Maxillofacial Surgery, Pécs, Dischka Gy. str. 5) Materials and methods: Tryptic digestion of salivary stones: grindedsalivary stones were extracted and denatured with urea, ammonium bicarbonate. Disulfide bonds were broken with dithiothreitol (DTT) and carbamidometilation was made with iodoacetamide. The reaction was stopped with β –mercaptoethanol and tryptic digestion took place overnight. Matrix: Alpha-cyano-4-hydroxycinnamic acid (CHCA ) was applied with „dried droplet” method in 1 to1 ratio with the samples Mass spectrometry: Bruker Autoflex II MALDI TOF/TOF (Bruker, Bremen, Germany) instrument was used in positive reflectron mode. Spectra of peptides were the sum of 1500 shots (30 shots added, each means 50 laser shots per spot), external calibration has been implemented. The detection was made in 800-4000 m/z range. Peptide mass fingerprints were collected and the results were sent to the Mascot Database. Data processing was executed with Flex Analysis software packages (version: 2.4.). For the insilico digestion Sequence Editor Software (Bruker Daltonics, Bremen, Germany) was used with the following criteria: 1. All cysteines were supposed to be treated with iodoacetamide 2. Monoisotopic masses were allowed 3. The maximum number of missed cleavage sites was two. Peptide mass fingerprints were collected from salivary stone extracts.Significant result is represented for human defensin protein HNP-3 chain A, in 50 ppm mass tolerance range. The four masses (986.501, 1142.570, 1273.579 and 1117.501) which were found to be identical from the PMF are the masses of four overlapping peptides. The mass error values are very low (8, 35, 25 and 8 ppm, as long as the mass tolerance range was 50 ppm). The sequence coverage is quite high; 63 % (19 amino acids are found from the 30) and the relatively high score value (79) also confirm the probability of our finding. Defensins are highly basic cationic peptides that are important components of the innate and adaptive immune response pathways. Cells of the immune system contain these peptides to assist in killing phagocytized bacteria, for example in neutrophyle granulocytes and almost all epithelial cells. Most defensins function by binding to microbial cell membrane, and once embedded, forming pore-like membrane defects that allow efflux of essential ions and nutrients. The MS/MS spectrum of the peptide with mass of 986.496 The MS/MS spectrum of the peptide with mass of 1142.585 Human defensin protein HNP-3 chain A was proven to be present in sialoliths. The PMF mass search from Mascot Database gave significant result. The small mass errors, the high sequence coverage and high score value prove our finding. MS/MS measurements were completed from the intensive masses and sequencing gave the same results, that verifies the presence of defensin in the salivary stones. The protein was isolated from the nucleus surroundings and peripheric layers, as long as in the central core no protein structures were detected. Previously made microscopical structure analysis (made with electronmicroscopy (EM), scanning electronmicroscopy (SEM), micro computed tomography (microCT) (results are not described here) showed that organic structures (collagen fibers, bacterial structures, epithelial cells, red blood cells) are visible only on the peripheral parts. Our MALDI results affirmed the structural observation, that protein structures are identifiable in salivary stones, however not in the central core. Summary: The organic core theory that stone formation is starting on a central protein structure (e.g.: epithelial cell, bacterial fragment) was contradicted in this study. These results can strengthen the findings of Kasaboglu et al ( 2004, 62:1253-1258, JOMS).