Dataset Information

Spleen_LTQ_ORBI_VELOS

ABSTRACT: Spleen, LTQ Orbitrap Velos

REANALYSIS of: PXD000479

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Danio Rerio

SUBMITTER: Kelkar DS, et al.

PROVIDER: PAe004371 | PeptideAtlas | 2014-12-31

REPOSITORIES: PeptideAtlas

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Publications

Annotation of the zebrafish genome through an integrated transcriptomic and proteomic analysis.

Kelkar Dhanashree S DS Provost Elayne E Chaerkady Raghothama R Muthusamy Babylakshmi B Manda Srikanth S SS Subbannayya Tejaswini T Selvan Lakshmi Dhevi N LD Wang Chieh-Huei CH Datta Keshava K KK Woo Sunghee S Dwivedi Sutopa B SB Renuse Santosh S Getnet Derese D Huang Tai-Chung TC Kim Min-Sik MS Pinto Sneha M SM Mitchell Christopher J CJ Madugundu Anil K AK Kumar Praveen P Sharma Jyoti J Advani Jayshree J Dey Gourav G Balakrishnan Lavanya L Syed Nazia N Nanjappa Vishalakshi V Subbannayya Yashwanth Y Goel Renu R Prasad T S Keshava TS Bafna Vineet V Sirdeshmukh Ravi R Gowda Harsha H Wang Charles C Leach Steven D SD Pandey Akhilesh A

Molecular & cellular proteomics : MCP 20140724 11

Accurate annotation of protein-coding genes is one of the primary tasks upon the completion of whole genome sequencing of any organism. In this study, we used an integrated transcriptomic and proteomic strategy to validate and improve the existing zebrafish genome annotation. We undertook high-resolution mass-spectrometry-based proteomic profiling of 10 adult organs, whole adult fish body, and two developmental stages of zebrafish (SAT line), in addition to transcriptomic profiling of six organs ...[more]

PMID: 25060758

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Project description:To identify low abundance autocrine growth factors in CHO cell conditioned media, we utilized a label-free shotgun proteomics approach. CHO cell conditioned media were harvested from fed batch bioreactors and concentrated using methanol/chlorofrom precipitation. Proteins in the samples were then subjected to proteolysis with trypsin, and then subjected to primary fractionation using a SCX column, followed by RP liquid chromatography MS (LC-MS) with a LTQ Orbitrap Velos instrument using the data dependent acquisition (DDA) method. The MS system was set up and run with a method which enabled fast acquisitions of high quality peptide precursor and fragment ion data, with the desired precursor mass accuracy of ±5 ppm. For the LTQ Orbitrap Velos MS, the data-dependent MS/MS analytical workflow in positive ion mode was used. Each precursor survey scan (m/z: 300 to 1800) by the Orbitrap mass analyzer (resolution = 60,000 FWHM) was linked to 10 MS/MS events using the 2D ion trap CID approach, with dynamic ion exclusion set at 60 s. This value was determined based on the observed mean peptide chromatographic peak width. All other instrument parameters were set up according to the manufacturer’s suggested values for complex peptide samples. The nano-ESI source was fitted with a 30-µm stainless steel nano-bore emitter (Thermo Fisher Scientific) with 1.7 kV applied near the tip. Raw data files from the LTQ Orbitrap Velos MS were processed using the Proteome Discoverer 1.3 software (Thermo Fisher Scientific). The LC-MS data were searched against the human (Homo sapiens; UniProtKb, updated in August 2012, 45 848 entries), mouse (Mus musculus; UniProtKb, updated in August 2012, 31 528 entries) and chinese hamster (Cricetulus griceus; UniprotKb, updated in August 2012, 24 609 entries) protein databases using the Sequest search engine for the LTQ Orbitrap Velos LC-MS data, assuming tryptic digestion with precursor ions to fall within 10 ppm of projected m/z values and a fragment ion mass tolerance of 0.5 m/z. The specified search parameters were carbamidomethylation of cysteine as fixed modification, oxidation of methionine as dynamic modification and a maximum of two missed cleavage events. Reverse database searching resulted in a specific false discovery rate (FDR) of 1% at the peptide and protein level.

Dataset Information

Spleen_LTQ_ORBI_VELOS

Publications

Annotation of the zebrafish genome through an integrated transcriptomic and proteomic analysis.

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