PXD005421

PXD005421 is an original dataset announced via ProteomeXchange.

Title	Large-scale filter-aided sample preparation method for the analysis of the ubiquitinome
Description	Protein ubiquitination regulates key cellular functions including protein homeostasis and signal transduction. The digestion of ubiquitinated proteins with trypsin yields a glycine-glycine remnant bound to the modified lysine residue (K-ε-GG) that can be recognized by specific antibodies for immunoaffinity purification (IAP) and subsequent identification of ubiquitination sites by mass spectrometry. Previous ubiquitinome studies based on this strategy have consistently digested milligram amounts of protein as starting material using in-solution digestion protocols prior to K-ε-GG enrichment. Filter-aided sample preparation (FASP) surpasses in-solution protein digestion in cleavage efficiency but its performance has thus far been shown for digestion of sample amounts on the order of micrograms. Because cleavage efficiency is pivotal in the generation of the K-ε-GG epitope recognized during IAP, here we developed a large-scale FASP method (LFASP) for digestion of milligram amounts of protein and evaluated its applicability to the study of the ubiquitinome. Our results demonstrate that LFASP-based tryptic digestion is efficient, robust, reproducible and applicable to the study of the ubiquitinome. We benchmark our results with state-of-the-art ubiquitinome studies and show an ~3-fold reduction in the proportion of miscleaved peptides with the method presented here. Beyond ubiquitinome analysis, LFASP overcomes the general limitation in sample capacity of standard FASP-based protocols and can therefore be used for a variety of applications that demand a large(r) amount of starting material.
HostingRepository	PRIDE
AnnounceDate	2017-03-30
AnnouncementXML	Submission_2017-03-30_05:36:53.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Montserrat Carrascal
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	ubiquitination signature dipeptidyl lysine
Instrument	LTQ Orbitrap

Revision	Datetime	Status	ChangeLog Entry
0	2016-11-22 06:02:37	ID requested
⏵ 1	2017-03-30 05:36:54	announced

Casanovas A, Pinto-Llorente R, Carrascal M, Abian J, Large-Scale Filter-Aided Sample Preparation Method for the Analysis of the Ubiquitinome. Anal Chem, 89(7):3840-3846(2017) [pubmed]

curator keyword: Biological

submitter keyword: LFASP, ubiquitinome, proteome, post-translational modifications, portein digestion, large-scale

Joaquin Abian Moñux
contact affiliation	CSIC/UAB Proteomics Laboratory, IIBB-CSIC, Barcelona, Spain
contact email	joaquim.abian.csic@uab.cat
lab head
Montserrat Carrascal
contact affiliation	IIBB-CSIC
contact email	montserrat.carrascal.csic@uab.cat
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD005421
     1. Label: PRIDE project
     2. Name: Large-scale filter-aided sample preparation method for the analysis of the ubiquitinome