MassIVE MSV000080845

Imported Reanalysis Dataset Public PXD000705

Extending proteome coverage through an on-line 2D SCX-RP ultra-high pressure system

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Description

Ultra high pressure liquid chromatography (UHPLC) systems combined with state of the art mass spectrometers have pushed the limit of deep proteome sequencing to new heights making it possible to identify thousands of proteins in a single LC/MS experiment within a few hours. However, the relationship between gradient length and the number of proteins identified is not linear and as gradient time increases to several hours, performance gain diminishes. Proteome coverage can be extended using 2-dimensional chromatography, but traditionally this comes at the expense of sample losses and much longer analysis times. Here, we asked the question whether a fast and sensitive online 2D SCX-RP UHPLC-MS/MS workflow, could compete with the current 1D long gradient analysis, making total analysis time- versus proteome coverage and sample used as benchmark parameters. Our new automated 2D-LC/MS system is robust and easy to use, consisting of a homemade SCX column, a trap column and a 50 cm analytical EASY-Spray column. We benchmark the system using small amounts of a human cell lysate digest. The 2D SCX-RP UHPLC-MS/MS workflow allowed us to identify 56600 unique peptides and 5958 proteins in a total analysis time of just over 8 hours. On the same system a 1D RP UHPLC-MS/MS workflow gave only 22000 peptides and 4200 unique proteins in 6 hours analysis time, increases of 157% and 42% at the peptide and protein level, respectively. These and other data reported here reveal that with this fast online SCX-RP UHPLC-MS/MS workflow proteome coverage can be substantially extended without compromising too much analysis time and sample usage [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: 2D LC-MS/MS ; Proteomics

Contact

Principal Investigators:
(in alphabetical order) 		Albert J.R. Heck, Biomolecular Mass Spectrometry and Proteomics groups, Utrecht University, N/A
Submitting User: ccms

Publications

Marino F, Cristobal A, Binai NA, Bache N, Heck AJ, Mohammed S.
Characterization and usage of the EASY-spray technology as part of an online 2D SCX-RP ultra-high pressure system.
Analyst. 2014 Dec 21;139(24):6520-8.

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Identification Results
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Quantification Results
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When complete, the converted files will be available in the "ccms_peak" subdirectory of the dataset's FTP space (accessible via the "FTP Download" link to the right).
Number of distinct conditions across all analyses (original submission and reanalyses) associated with this dataset.

Distinct condition labels are counted across all files submitted in the "Metadata" category having a "Condition" column in this dataset.

"N/A" means no results of this type were submitted.
Number of distinct biological replicates across all analyses (original submission and reanalyses) associated with this dataset.

Distinct replicate labels are counted across all files submitted in the "Metadata" category having a "BioReplicate" or "Replicate" column in this dataset.

"N/A" means no results of this type were submitted.
Number of distinct technical replicates across all analyses (original submission and reanalyses) associated with this dataset.

The technical replicate count is defined as the maximum number of times any one distinct combination of condition and biological replicate was analyzed across all files submitted in the "Metadata" category. In the case of fractionated experiments, only the first fraction is considered.

"N/A" means no results of this type were submitted.
Originally identified proteins that were automatically remapped by MassIVE to proteins in the SwissProt human reference database.

"N/A" means no results of this type were submitted.
Number of distinct protein accessions reported across all analyses (original submission and reanalyses) associated with this dataset.

"N/A" means no results of this type were submitted.
Number of distinct unmodified peptide sequences reported across all analyses (original submission and reanalyses) associated with this dataset.

"N/A" means no results of this type were submitted.
Number of distinct peptide sequences (including modified variants or peptidoforms) reported across all analyses (original submission and reanalyses) associated with this dataset.

"N/A" means no results of this type were submitted.
Total number of peptide-spectrum matches (i.e. spectrum identifications) reported across all analyses (original submission and reanalyses) associated with this dataset.

"N/A" means no results of this type were submitted.
Number of distinct proteins quantified across all analyses (original submission and reanalyses) associated with this dataset.

Distinct protein accessions are counted across all files submitted in the "Statistical Analysis of Quantified Analytes" category having a "Protein" column in this dataset.

"N/A" means no results of this type were submitted.
Number of distinct proteins found to be differentially abundant in at least one comparison across all analyses (original submission and reanalyses) associated with this dataset.

A protein is differentially abundant if its change in abundance across conditions is found to be statistically significant with an adjusted p-value <= 0.05 and lists no issues associated with statistical tests for differential abundance.

Distinct protein accessions are counted across all files submitted in the "Statistical Analysis of Quantified Analytes" category having a "Protein" column in this dataset.

"N/A" means no results of this type were submitted.
This dataset may not contain all raw spectra data as originally deposited in PRIDE. It has been imported to MassIVE for reanalysis purposes, so its spectra data here may consist solely of processed peak lists suitable for reanalysis with most software.