Dataset Information

Identifcation of Adiponectin and Evidence for Post-Translational Modifications in the Bottlenose Dolphin (Tursiops truncatus)

ABSTRACT: A Multiple Affinity Removal System (MARS) Human 14 spin cartridge was used to deplete a dolphin serum which was then digested with trypsin and analyzed without prior fractionation by nanoLC-MS/MS. These files are associated with "2011-9-25_Neely-DolphinSerum." Data analysis: raw data generated by the AB Sciex 5600 were converted to a peak list using the AB Sciex MS Data Converter (v. 1.1 beta, July 2011). Protein identifications were made using Mascot (v. 2.3.02) searching against the Ensembl (release 64) turTru1 dolphin genome assembly protein database [16,598 sequences] and the common Repository of Adventitious Proteins database (cRAP; 2012.01.01; the Global Proteome Machine) using the following parameters: trypsin was selected as the enzyme and three missed cleavages were allowed; carbamidomethylation (Cys) was specified as a fixed modification; glucosylgalactosyl (Lys), oxidation (Met, Pro), and 2-succinyl (Cys) were specified as variable modifications; a precursor tolerance of 10 ppm and fragment ion tolerance of 0.5 Da; instrument type was ESI-QUAD-TOF. Additionally, a non-depleted dolphin sera that had been digested followed by quasi-fractionation using SPE, was analyzed by nano-LC-MS/MS which lead to an additional peptide identification, KGEPGESAYVYR, and these files are associated with "2012-12-12 Dolphin IDA".

INSTRUMENT(S): TripleTOF 5600

ORGANISM(S): Tursiops Truncatus (atlantic Bottle-nosed Dolphin) (delphinus Truncatus)

SUBMITTER: Benjamin Neely

PROVIDER: PXD000431 | Pride | 2013-09-23

REPOSITORIES: Pride

ACCESS DATA

Publications

Ratiometric Measurements of Adiponectin by Mass Spectrometry in Bottlenose Dolphins (Tursiops truncatus) with Iron Overload Reveal an Association with Insulin Resistance and Glucagon.

Neely Benjamin A BA Carlin Kevin P KP Arthur John M JM McFee Wayne E WE Janech Michael G MG

Frontiers in endocrinology 20130920

High molecular weight (HMW) adiponectin levels are reduced in humans with type 2 diabetes and insulin resistance. Similar to humans with insulin resistance, managed bottlenose dolphins (Tursiops truncatus) diagnosed with hemochromatosis (iron overload) have higher levels of 2 h post-prandial plasma insulin than healthy controls. A parallel reaction monitoring assay for dolphin serum adiponectin was developed based on tryptic peptides identified by mass spectrometry. Using identified post-transla ...[more]

PMID: 24065958

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Project description:SUMO modification of proteins (sumoylation) is essential for mitotic progression from yeast to humans, but only a limited number of sumoylated proteins with functions in mitosis have been discovered. Vertebrates express three SUMO paralogs, SUMO-1, SUMO-2 and SUMO-3, but only SUMO-2 and -3 are chromosome-associated in mitosis. In this study, we used chromosome spreads to more precisely define the localization of endogenous SUMO-2 and -3 to the centromere as well as the chromosome protein scaffold. Furthermore, we developed methodologies for the immunopurification of endogenous SUMO-2 and -3 modified proteins from cell extracts. Using LC-MS/MS, we analyzed proteins immunopurified from mitotic chromosome fractions and G0 nuclear fractions. We identified 296 putative sumoylated proteins, with 138 being modified specifically in mitosis. We identified proteins known to localize to the centromere and kinetochore and the chromosome protein scaffold, consistent with SUMO-2/3 localization. Furthermore, we demonstrate that utilizing cell synchronization and fractionation allowed for the discovery of low abundance sumoylated proteins that are not detected in large asynchronous analyses. Our results provide a foundation for further characterization of the roles of sumoylation in regulating diverse aspects of chromosome segregation in mitosis. Thermo .raw files were uploaded to the ProHits (Liu et al, 2010) analytical suite and converted to .mzXML format using ReAdW software. Data were searched using X!Tandem (Craig & Beavis, 2004) against human ORFs (RefSeq v45). Search parameters specified a parent MS tolerance of +/- 15ppm, and an MS/MS tolerance of 0.4 Da, with up to two missed cleavages for trypsin. Oxidation of methionine and tryptophan, ubiquitylation of lysine, and alkylation of cysteine (by NEM) were allowed as variable modifications. Statistical validation of the results was performed using Peptide Prophet and Protein Prophet (Keller et al, 2002; Nesvizhskii et al, 2003) as part of the trans-proteomic pipeline. For each search, the Protein Prophet probability at a 1% false discovery rate was used as a cutoff value to generate SAINT-compatible input files. SAINT parameters were as follows: 5000 iterations, low mode Off (0), minFold 1 and normalization (1) (Choi et al, 2011). SAINT cutoff values of 0.75 were used to generate a high-confidence list of protein identifications.

Project description:Before stimulation, the cells were washed and the culture media was changed to RPMI-1640. After stimulation, cell culture media were collected, concentrated, and the proteins were precipitated with 2-D Clean-Up Kit (GE Healthcare) followed by protein alkylation, trypsin digestion and iTRAQ labelling of the resulting peptides according to manufacturer´s instructions (Applied Biosystems). Control sample was labeled with 114, LPS-stimulated with 115, Curdlan-stimulated with 116, and GBY-stimulated with 117 isobaric tag. After labelling the samples were pooled and dried, and the peptides were fractionated by strong cation exchange chromatography using an Ettan HPLC system (Amersham Biosciences) connected to a PolySULFOETHYL A column. Each SCX-fraction containing labelled peptides was analyzed twice with nano-liquid chromatography-tandem mass spectrometry using Ultimate 3000 nano-liquid chromatograph (Dionex) and QSTAR Elite hybrid quadrupole time-of-flight mass spectrometer (Applied Biosystems / MDS Sciex) with nano-ESI ionization as previously described (Lietzén et al., 2011). MS data were acquired automatically using Analyst QS 2.0 software. Protein identification and relative quantitation were performed with ParagonTM search algorithm (Shilov et al., 2007) using ProteinPilot 2.0 interface (AB Sciex). Data files from both technical replicates of an iTRAQ sample set were processed together. Database searching was done against UniProt human database (version 2008-01-28 with 20330 human sequences) and ´decoy` database (the reverse amino acid sequence for false discovery rate estimation). The search criteria were: cysteine alkylation with MMTS, trypsin digestion, biological modifications allowed, thorough search and detected protein threshold of 95% confidence (Unused ProtScore > 1.3). Importantly, automatic bias correction was not used in quantitation. The false discovery rates were calculated as previously described (Elias and Gygi, 2007) and were 1.4% and 2% for the two biological replicates.

Dataset Information

Identifcation of Adiponectin and Evidence for Post-Translational Modifications in the Bottlenose Dolphin (Tursiops truncatus)

Publications

Ratiometric Measurements of Adiponectin by Mass Spectrometry in Bottlenose Dolphins (Tursiops truncatus) with Iron Overload Reveal an Association with Insulin Resistance and Glucagon.

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