PXD012556

PXD012556 is an original dataset announced via ProteomeXchange.

Title	Deep profiling and custom databases improve detection of proteoforms generated by alternative splicing
Description	Alternative pre-mRNA splicing has long been proposed to greatly contribute to proteome complexity. However, the extent to which mature mRNA isoforms are successfully translated into protein remains controversial. Here, we used high-throughput RNA sequencing and mass spectrometry (MS)-based proteomics to better evaluate the translation of alternatively spliced mRNAs. To increase proteome coverage and improve protein quantitation, we optimized cell fractionation and sample processing steps at both the protein and peptide level. Furthermore, we generated a custom spectral library trained on analysis of RNA-Seq data with MAJIQ, an algorithm optimized to detect differential and unannotated junction usage for a given splice site. Using this custom library to match against tandem mass spectra acquired by data independent acquisition (DIA), we improved identification of splicing-derived proteoforms by ~30% as compared to use of the SwissProt database alone. Moreover, our increased depth and detection of proteins allowed us to track changes in the transcriptome and proteome induced by T cell stimulation, as well as fluctuations in protein sub-cellular localization. In sum, our data here confirms that use of generic databases in proteomic studies under-estimates the number of spliced mRNA isoforms that are translated into protein and provides a workflow that improves isoform detection in large-scale proteomic experiments.
HostingRepository	PRIDE
AnnounceDate	2024-10-22
AnnouncementXML	Submission_2024-10-22_04:52:40.136.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Laura Agosto
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	monohydroxylated residue; iodoacetamide derivatized residue
Instrument	Orbitrap Fusion; Q Exactive

Revision	Datetime	Status	ChangeLog Entry
0	2019-01-29 03:47:44	ID requested
1	2019-10-14 01:22:39	announced
2	2019-12-11 06:58:39	announced	2019-12-11: Updated publication reference for PubMed record(s): 31727681.
⏵ 3	2024-10-22 04:52:48	announced	2024-10-22: Updated project metadata.

10.1101/gr.248435.119;

Agosto LM, Gazzara MR, Radens CM, Sidoli S, Baeza J, Garcia BA, Lynch KW, Deep profiling and custom databases improve detection of proteoforms generated by alternative splicing. Genome Res, 29(12):2046-2055(2019) [pubmed]

curator keyword: Biological

submitter keyword: Human, proteoforms, alternative splicing, nanoLC-MS/MS, proteome, Jurkat T cells, RNA-Seq

Benjamin A. Garcia
contact affiliation	Epigenetics Institute, Department of Biochemistry and Biophysics, Univeristy of Pennsylvania
contact email	bgarci@pennmedicine.upenn.edu
lab head
Laura Agosto
contact affiliation	University of Pennsylvania Perelman School of Medicine
contact email	lauag@pennmedicine.upenn.edu
dataset submitter

Dataset FTP location NOTE: Most web browsers have now discontinued native support for FTP access within the browser window. But you can usually install another FTP app (we recommend FileZilla) and configure your browser to launch the external application when you click on this FTP link. Or otherwise, launch an app that supports FTP (like FileZilla) and use this address: ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/10/PXD012556

PRIDE project URI

• PRIDE
  1. PXD012556
     1. Label: PRIDE project
     2. Name: Deep profiling and custom databases improve detection of proteoforms generated by alternative splicing