Mass spectrometry identification of granins and other proteins secreted by neuroblastoma cells

Wojciech Rozek 0 Malgorzata Kwasnik 0 Janusz Debski 0 Jan F. Zmudzinski 0 0 J. Debski Mass Spectrometry Laboratory, Institute of Biochemistry and Biophysics , PAS, Warsaw, Poland We used mass spectrometry-based protein identification to determine the presence of granins and other proteins in the mouse neuroblastoma secretome. We detected polypeptides derived from four members of the granin family: chromogranin A, chromogranin B, secretogranin III, and VGF. Many of them are derived from previously described biologically active regions; however, for VGF and CgB, we detected peptides not related to known bioactivities. Along with granins, we identified 115 other proteins secreted by mouse neuroblastoma cells, belonging to different functional categories. Fifty-six out of 119 detected proteins possess the signal fragments required for translocation into endoplasmic reticulum. Sequences of remaining 63 proteins were analyzed using SecretomeP algorithm to determine probability of nonclassical secretion. Identified proteins are involved in the regulation of cell cycle, proliferation, apoptosis, angiogenesis, proteolysis, and cell adhesion. - Over the last few years, there has been a growing interest in the study of cancer secretome comprising all the proteins that can be identified in the intestinal fluid of the tumor mass in vivo which play a key role in the signaling, communication, and migration of cells [24, 33]. The term of secretome was introduced by Tjalsma in genome-based studies of Bacillus subtilis proteins [51]. Currently, the secretome studies include the proteins secreted via classical and nonclassical pathways but also shed from the surface of living cells [33]. The cell culture secretome can also be a suitable tool for investigating proteins released in vivo by tumors and used to identify putative tumor markers [9]. Neuroblastoma is the most common extracranial solid tumor of the sympathetic nervous system occurring in childhood. This neuroendocrine tumor secretes a range of proteins, which could serve as the potential biomarkers for diagnosis and monitoring of the treatment or disease progression [11, 46]. Several serum prognostic factors, such as neuron specific enolase, ferritin, and chromogranin A (CgA) have been used to predict neuroblastoma progression. CgA is currently the best available biomarker for the diagnosis of neuroendocrine tumors [17, 22, 55]. The granin family comprises nine members including CgA and CgB, secretogranin (Sg) II, III, IV (HISL-19), V (7B2), VI (NESP55), VII (VGF), and proSAAS [15, 16, 18, 56]. Potential utility of CgB, SgII, and VGF nerve growth factor-inducible protein (VGF) as biomarkers of neurological and psychiatric disorders has been described [6]. The expression patterns of granin-derived peptides seem to play an important role in differentiating between some benign and malignant neuroendocrine tumor types [39]. Granins are the main soluble proteins found in many neuroendocrine cells and in some neurons. They are present in large dense-core secretory vesicles and secreted during regulated exocytosis. Granins regulate the storage of catecholamines and ATP, exhibit pHbuffering capacities and thus they help to concentrate soluble products for secretion [7, 18, 32]. Their sequences contain pairs of basic amino acids and monobasic residues that are the potential cleavage sites for proteases. The granin-derived peptides fulfill autocrine and paracrine hormonal activities. Their relative abundance, functional significance, and secretion into the CSF or saliva and the general circulation made granin peptides tractable targets as biomarkers for many diseases of neuronal and endocrine origin [6]. We used mass spectrometry-based protein identification to determine the presence of the granin and other protein-derived peptides in the neuroblastoma secretome. This approach could deliver new information regarding neuroblastoma metabolism and new potential biomarkers of the disease. Material and methods Sample preparation The mouse neuroblastoma cell line NEURO-2A was cultured in Eagles medium with 10 % fetal bovine serum. Oneday-old cultures were washed twice with PBS and the serum-free medium was applied. After 24 h culture, media were collected and centrifuged at 3,000g for 30 min. The supernatants were concentrated on centrifugal filters with the molecular weight cutoff of 3 kDa (Millipore, UFC900324). Proteins were precipitated using 5 volumes of cold acetone (20 C) and samples were centrifuged at 12,000g for 10 min at 4 C. Subsequently, pellets were resuspended in 8 M urea and diluted with 25 mM ammonium bicarbonate. Proteins were reduced with 10 mM DTT for 30 min at 57 C and alkylated with 50 mM iodoacetamide for 45 min at room temperature (RT) in a dark. Then samples were treated with 50 mM DTT for 45 min at RT. Seventy micrograms of protein was used for tryptic digestion and protein identification. Solubilized proteins were digested overnight with sequencing grade modified trypsin (Promega, V5111, 0.01 g per 1 g of protein) and the reaction was quenched by adding 0.01 % trifluoroacetic acid. Mass spectrometry and data analysis Digested peptides were applied to a RP-18 trapping column (nanoACQUITY UPLC Symmetry C18 Trap, Waters) using 0.1 % trifluoroacetic acid mobile phase, and then transferred on to a HPLC RP-18 column (nanoACQUITY UPLC BEH C18 Column, Waters) using an acetonitrile gradient (030 % in 0.1 % formic acid) for 150 min at a flow rate of 200 nL/min. The column outlet was directly coupled to the ion source of the Ion Cyclotron Resonance spectrometer (LTQ61 FTICR, Thermo Electron). For protein identification, a series of three LC/MS runs were carried out on each sample, with the spectrometer running in data-dependent MS-to-MS/MS switch mode. Each run covered one of sectors of m/z values: 300600, 500800, 7002000. The parent and product ions lists for the database search were prepared by merging acquired raw files with Mascot Distiller software followed by Mascot Search Engine (Matrix Science, London, UK) against the NCBInr and IPIMouse database. Search parameters for precursor and product ions mass tolerance were 30 ppm and 0.8 Da, respectively. The other search parameters were as follows: enzyme specificity was set up to trypsin cleavage and variable modification of cysteine carbamidomethylation and methionine oxidation. Peptides with Mascot score exceeding the threshold value corresponding to <5 % false positive rate, calculated by Mascot procedure, were considered to be positively identified. At least two peptides per protein with score above the threshold were required for identification. The whole experiment was performed twice, using two biological replicates. Functional categorization of proteins was performed using Protein Analysis Through Evolutionary Relationship system (PANTHER, http:// www.pantherdb.org/) [36] and Gene Ontology (GO) classification [3]. We determined the presence of the signal peptide (...truncated)