Dataset Information

Shotgun Imaging Analyzer Gastric Cancer

ABSTRACT: The data discloses mass spectra of a gastric cancer biopsy sectioned into ten parts, each one subjected to a four step MudPIT analysis (i.e., increasing KCL concentrations of 85, 150, 250, and 400 mM). The biopsy was acquired from an area along the stomach that included tumor and resection margin, during the operation procedures on a male patient. Briefly, the tumor was located in the gastric antrum and the resection margin was macroscopically defined during the surgery as a 10 cm rim of healthy-looking tissue surrounding the tumor. The histological type was determined to be adenocarcinoma and classified as T4. Data analysis: The ProLuCID search engine (v 1.3) was used to compare experimental tandem mass spectra against those theoretically generated from our sequence database and select the most likely peptide spectrum matches (PSMs). Briefly, the search was limited to fully and semi-tryptic peptide candidates and imposed carbamidomethylation and oxidation of methionine as fixed and variable modification, respectively. The search engine accepted peptide candidates within a 50-ppm tolerance from the measured precursor m/z and used the XCorr and Z-Score as the primary and secondary search engine scores, respectively. The validity of the PSMs was assessed using the Search Engine Processor (SEPro, v 2.1.0.23). Identifications were grouped by charge state (+2 and > +3) and then by tryptic status (fully tryptic, semi-tryptic), resulting in four distinct subgroups. For each result, the ProLuCID XCorr, DeltaCN, and ZScore values were used to generate a Bayesian discriminator. Additionally, a minimum sequence length of six amino acid residues was required. Results were post-processed to only accept PSMs with less than 5 ppm and proteins supported by two or more independent evidences (e.g., identification of a peptide with different charge states, a modified and a non-modified version of the same peptide, or two different peptides).

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Paulo Carvalho

PROVIDER: PXD000584 | Pride | 2013-12-05

REPOSITORIES: Pride

ACCESS DATA

Publications

Exploring the proteomic landscape of a gastric cancer biopsy with the shotgun imaging analyzer.

Aquino Priscila Ferreira PF Lima Diogo Borges DB de Saldanha da Gama Fischer Juliana J Melani Rafael Donadélli RD Nogueira Fabio C S FC Chalub Sidney R S SR Soares Elzalina R ER Barbosa Valmir C VC Domont Gilberto B GB Carvalho Paulo C PC

Journal of proteome research 20131127 1

Accessing localized proteomic profiles has emerged as a fundamental strategy to understand the biology of diseases, as recently demonstrated, for example, in the context of determining cancer resection margins with improved precision. Here, we analyze a gastric cancer biopsy sectioned into 10 parts, each one subjected to MudPIT analysis. We introduce a software tool, named Shotgun Imaging Analyzer and inspired in MALDI imaging, to enable the overlaying of a protein's expression heat map on a tis ...[more]

PMID: 24283986

Similar Datasets

Project description:In recent years, high-throughput technologies have contributed to development a more precise picture of the human proteome. However, 2,129 proteins remain listed as missing proteins (MPs) in the newest neXtProt release (2019-02). The main reasons for MPs are a low abundance, low-molecular-weight (LMW), unexpected modifications, membrane characteristics, etc. Moreover, more than 50% of the MS/MS data have not been successfully identified in shotgun proteomics. Open-pFind, an efficient open search engine, recently released by the pFind group in China, presents an opportunity to identify these buried MPs in complex samples. Proteins and potential MPs were identified using Open-pFind and three other search engines to compare their performance and efficiency with three large-scale datasets digested by different enzymes. Our results demonstrated that Open-pFind identified 29.9-47.5% more peptide-spectrum matches (PSMs), 48.0-63.9% more peptides sequences (with modifications) and 22.7-38.1% more peptide sequences (regardless of modifications) than those identified by the second-best search engine. As a result, Open-pFind detected 7.5-19.3% more candidate MPs than those by the second-best search engine. In total, 5 (PE2) of the 150 candidate MPs identified by Open-pFind were verified from two unique peptides containing more than 9 amino acids (AA) by using spectrum theoretical prediction with pDeep, and synthesized peptide matching with pBuild, after spectrum quality analysis, isobaric post-translational modification, and single amino acid variant (SAAV) filtering. These five verified MPs can be ranked in the PE1 level. In addition, three other candidate MPs were verified with two unique peptides (one peptide containing more than 9 AA) and the other containing only 8 AA), which were slightly lower than the criteria listed by C-HPP, and required additional verification information. More importantly, unexpected modifications were detected in these MPs. Another 141 MPs were listed as candidates, but required additional verification information.

Project description:B. japonicum cells were grown either in minimal medium (BVM) with L-arabinose as carbon and energy source or in complete medium (PSY) containing L-arabinose. Proteins extracted from three replicates of B. japonicum cells grown in complex and minimal medium with L-arabinose until mid-exponential phase were separated on a Tris-HCl polyacrylamide gel. After reduction and carbamidomethylation, the proteins were digested with trypsin (Promega, Madison, WI, USA), and the resulting peptides were separated by RP-HPLC and analyzed by a hybrid LTQ-Orbitrap XL mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) interfaced with a nanoelectrospray source. Mass spectra were further processed with an in-house processing pipeline. A total of 3,390 and 3,149 proteins were identified in complex and minimal medium respectively. We identified highly expressed products of candidate genes involved in the degradation of arabinose and suggest that glycolaldehyde is oxidized to glyoxylate and then reduced to glycerate through a glyoxylate carboligase and a tartronate semialdehyde reductase for assimilation and/or converted to oxalate and then oxidized to formate and CO2 through a formyl-CoA transferase (Frc) and an oxalyl-CoA decarboxylase (Oxc) for energy generation. Fragment ion mass spectra were extracted from Thermo RAW files using msconvert (ProteoWizard, version 3.0.3831), merged in one MGF file per gel and searched against a composite B. japonicum USDA 110 protein database (RefSeq NC_004463.1, July 22, 2013) containing 256 common contaminants (e.g. human keratin, trypsin) with the search engine MS-GF+ (MS-GFDB v7747) for a match to fully-tryptic and semi-tryptic peptides with up to two missed cleavage sites with a mass tolerance of 25ppm and with oxidation (M), deamidation (NQ), methylation (DE) as variable modifications and carbamidomethylation (C) as fixed modification. Using the decoy option of MS-GF+, we filtered the list of peptide spectrum matches (PSMs) to an estimated overall FDR of 0.2%, classified the PSMs with PeptideClassifier, and only further considered peptides (tryptic or semi-tryptic) that unambiguously imply one bacterial protein sequence. The FDR at the peptide level amounted to about 1.5%, that at the protein level to about 2.5% when requiring two peptides or three spectra for a protein identification.

Dataset Information

Shotgun Imaging Analyzer Gastric Cancer

Publications

Exploring the proteomic landscape of a gastric cancer biopsy with the shotgun imaging analyzer.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure