PXD005650 is an
original dataset announced via ProteomeXchange.
Dataset Summary
Title | Highly reproducible improved label-free quantitative analysis of cellular phosphoproteome by optimization of LC-MS/MS gradient and analytical column construction |
Description | Expanding the sequencing depth of the peptides showing a statistically significant quantitative change arising from a biological stimulation is critical. Here we demonstrate that optimization of LC gradient and analytical column construction can reveal over 30,000 unique peptides and 23,000 phosphopeptides at high confidence. The quantitative reproducibility of different analytical workflows was evaluated by comparing the phosphoproteome of CD3/4 stimulated and unstimulated T-cells as a model system. A fritless, 50 cm-long column packed with 1.9 µm particles operated with a standard pressure HPLC significantly improved the sequencing depth 51% and decreased the selected ion chromatogram peak spreading. Most importantly, under the optimal workflow we observed an improvement of 330% in detection of significantly changed phosphopeptides in the stimulated cells compared with the other workflows. The discovery power of the optimized column configuration was illustrated by identification of significantly altered phosphopeptides harboring novel sites from proteins previously established as important in T cell signaling including A-Raf, B-Raf, c-Myc, CARMA1, Fyn, ITK, LAT, NFAT1/2/3, PKCα, PLCγ1/2, RAF1, and SOS1. Taken together, our results revealed a simple column fabrication methodology that provides an inexpensive improvement for single-run LC-MS/MS analysis to optimize peptide sequencing depth, dynamic range, sensitivity, and label free quantitative reproducibility. |
HostingRepository | PRIDE |
AnnounceDate | 2018-10-26 |
AnnouncementXML | Submission_2018-10-26_11:11:45.xml |
DigitalObjectIdentifier | |
ReviewLevel | Peer-reviewed dataset |
DatasetOrigin | Original dataset |
RepositorySupport | Unsupported dataset by repository |
PrimarySubmitter | Arthur Salomon |
SpeciesList | scientific name: Homo sapiens (Human); NCBI TaxID: 9606; |
ModificationList | phosphorylated residue; iodoacetamide derivatized residue |
Instrument | Q Exactive |
Dataset History
Revision | Datetime | Status | ChangeLog Entry |
0 | 2017-01-03 07:43:52 | ID requested | |
⏵ 1 | 2018-10-26 11:11:46 | announced | |
Publication List
Ahsan N, Belmont J, Chen Z, Clifton JG, Salomon AR, Highly reproducible improved label-free quantitative analysis of cellular phosphoproteome by optimization of LC-MS/MS gradient and analytical column construction. J Proteomics, 165():69-74(2017) [pubmed] |
Keyword List
curator keyword: Technical, Biological |
submitter keyword: Human, T cell, phosphoproteomics, q exactive, acute leukemia, label free |
Contact List
Arthur Robert Salomon |
contact affiliation | Brown University, Molecular Biology, Cell Biology, Biochemistry |
contact email | art@drsalomon.com |
lab head | |
Arthur Salomon |
contact affiliation | Brown University |
contact email | art@drsalomon.com |
dataset submitter | |
Full Dataset Link List
Dataset FTP location
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PRIDE project URI |
Repository Record List
[ + ]
[ - ]
- PRIDE
- PXD005650
- Label: PRIDE project
- Name: Highly reproducible improved label-free quantitative analysis of cellular phosphoproteome by optimization of LC-MS/MS gradient and analytical column construction