PXD005650

PXD005650 is an original dataset announced via ProteomeXchange.

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

Revision	Datetime	Status	ChangeLog Entry
0	2017-01-03 07:43:52	ID requested
⏵ 1	2018-10-26 11:11:46	announced

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]

curator keyword: Technical, Biological

submitter keyword: Human, T cell, phosphoproteomics, q exactive, acute leukemia, label free

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

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PRIDE project URI

• PRIDE
  1. PXD005650
     1. Label: PRIDE project
     2. Name: Highly reproducible improved label-free quantitative analysis of cellular phosphoproteome by optimization of LC-MS/MS gradient and analytical column construction