PXD000421

PXD000421 is an original dataset announced via ProteomeXchange.

Title	PAPE - Pre-fractionation assisted phosphoprotein enrichment, a novel approach for phosphoproteomic analysis of green tissues from plants
Description	Phosphorylation is an important post-translational protein modification with regulatory roles in diverse cellular signaling pathways. Despite recent advances in mass spectrometry, the detection of phosphoproteins involved in signaling is still challenging, as protein phosphorylation is typically transient and/or occurs at low levels. In green plant tissues, the presence of highly abundant proteins, such as the subunits of the RuBisCO complex, further complicates phosphoprotein analysis. Here, we describe a simple, but powerful, method, which we named prefractionation-assisted phosphoprotein enrichment (PAPE), to increase the yield of phosphoproteins from Arabidopsis thaliana leaf material. The first step, a prefractionation via ammonium sulfate precipitation, not only depleted RuBisCO almost completely, but, serendipitously, also served as an efficient phosphoprotein enrichment step. When coupled with a subsequent metal oxide affinity chromatography (MOAC) step, the phosphoprotein content was highly enriched. The reproducibility and efficiency of phosphoprotein enrichment was verified by phospho-specific staining and, further, by mass spectrometry, where it could be shown that the final PAPE fraction contained a significant number of known and additionally novel (potential) phosphoproteins. Hence, this facile two-step procedure is a good prerequisite to probe the phosphoproteome and gain deeper insight into plant phosphorylation-based signaling events. MS raw data were searched against an A. thaliana protein database based on The Arabidopsis Information Resource (TAIR) 10 with the Proteome Discoverer 1.3 using an in-house Mascot server (precursor mass tolerance: 7 ppm; fragment mass tolerance: 0.8 Da; missed cleavages: 2). Carbamidomethylation of cysteine was set as a static modification. Variable modifications were oxidation (Methionine), acetylation (protein N-terminus), deamidation (Asparagine/Glutamine) and phosphorylation (Serine/Threonine). Further data evaluation was carried out with the Scaffold software (Version 3.3, Proteome Software Inc., Portland, OR, USA), Proteome Discoverer 1.3 with phosphoRS 1.0 (Thermo Fisher Scientific) and DanteR [30] for total protein content. Phosphopeptides were identified with the Proteome Discoverer 1.3 software, which includes the phosphoRS 1.0 algorithm (Thermo Fisher Scientific) for phospho-site mapping. A false discovery rate (FDR) was calculated by searching a ―decoy‖ database containing all the target database sequences in reverse order. Peptide-spectrum match (PSM) was set at a q-value <0.05 (i.e., a corrected significance threshold employing the Benjamini-Hochberg FDR procedure to control for a family-wise error rate). Protein grouping was enabled.
HostingRepository	PRIDE
AnnounceDate	2014-07-25
AnnouncementXML	Submission_2014-07-25_01:34:39.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Ines Lassowskat
SpeciesList	scientific name: Arabidopsis thaliana (Mouse-ear cress); NCBI TaxID: 3702;
ModificationList	deamidated residue; monohydroxylated residue; acetylated residue; iodoacetamide derivatized residue; phosphorylated residue
Instrument	LTQ Orbitrap Velos

Revision	Datetime	Status	ChangeLog Entry
0	2013-08-19 01:49:23	ID requested
1	2014-01-28 05:37:47	announced
⏵ 2	2014-07-25 01:34:39	announced	Updated project metadata.

Dataset with its publication pending

submitter keyword: Arabidopsis thaliana, leaf, LC-MS, LTQ Orbitrap Velos, phosphoproteomics

Ines Lassowskat
contact affiliation	SEB
contact email	ines.lassowskat@ipb-halle.de
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD000421
     1. Label: PRIDE project
     2. Name: PAPE - Pre-fractionation assisted phosphoprotein enrichment, a novel approach for phosphoproteomic analysis of green tissues from plants