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PXD009021

DataSet Summary

  • HostingRepository: PRIDE
  • AnnounceDate: 2018-04-23
  • AnnouncementXML: Submission_2018-04-23_05:07:41.xml
  • DigitalObjectIdentifier: http://dx.doi.org/10.6019/PXD009021
  • ReviewLevel: Peer-reviewed dataset
  • DatasetOrigin: Original data
  • RepositorySupport: Supported dataset by repository
  • PrimarySubmitter: Clay Davis
  • Title: Proteomic Analysis of Human Liver Reference Material
  • Description: Recent advances in mass spectrometric (MS) instruments resulting in high mass resolution and high duty cycles have allowed of very deep coverage of proteomes. However, even with state of the art, high duty cycle instruments, the number of proteins identified with a conventional single liquid chromatography-tandem MS (LC/MS/MS) analysis is typically limited and fractionating protein samples prior to LC/MS/MS analysis is crucial for increasing both the analytical dynamic range and proteome coverage. In order to achieve near comprehensive identification of proteomes two-dimensional (2D) chromatography has been an invaluable tool. This first dimension is typically performed with ÁL/min flow and relatively large column inner diameters, which allow efficient pre-fractionation but typically require peptide amounts in the milligram range while the second dimension is typically performed with nL/min flow rates and capillary columns with small inner diameters. Typically, reverse phase liquid chromatography (RPLC) provides high peak resolution of peptides and achieves higher peak capacities than strong cation exchange chromatography (SCX) due to the faster chromatographic partitioning. Another advantage of RPLC is that the mobile phases used generate much cleaner samples for downstream analyses, while the incorporation of a desalting step for SCX in order to eliminated the high salt concentrations that can significantly decrease the analytical sensitivity of the MS analysis due to ionization problems. When operated at widely different pH values (e.g., 1st dimension pH 10 and 2nd dimension pH 3) 2D RPLC-RPLC provides separation orthogonality comparable to that of the combination of SCX-RPLC. The difference of separation selectivity between the low and high pH (HpH) RPLC comes from the changes in the charge distribution within peptide chains upon altering pH of the eluent. Herein we describe a method in which tryptic peptides are separated in the first dimension by HpH-RPLC and fractions are collected every 90 s over an 80 min gradient. For the second dimension, each of these fractions is individually run by the standard low pH RPLC method. The implementation of the HpH pre-fractionation allows for short second dimension gradients for bottom up LC/MS/MS and yields very deep (e.g. thousands of identifications) protein coverage at reasonable measurement time.
  • SpeciesList: scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
  • ModificationList: Deamidated; Oxidation; Acetyl; Oxidation; Gln->pyro-Glu
  • Instrument: Orbitrap Fusion Lumos

Dataset History

VersionDatetimeStatusChangeLog Entry
02018-02-23 03:31:05ID requested
12018-04-23 05:07:42announced

Publication List

  1. Dataset with its publication pending

Keyword List

  1. curator keyword: Technical, Biological
  2. submitter keyword: Human, Liver

Contact List

    Clay Davis, Ben Neely
    • contact affiliation: Chemical Sciences Division National Institute of Standards and Technology, USA
    • contact email: clay.davis@nist.gov
    • lab head:
    Clay Davis
    • contact affiliation: National Institute of Standards and Technology
    • contact email: clay.davis@nist.gov
    • dataset submitter:

Full Dataset Link List

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  2. PRIDE project URI
Repository Record List
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