PXD000381

PXD000381 is an original dataset announced via ProteomeXchange.

Title	Identification of SUMO-2/3-modified proteins associated with mitotic chromosomes
Description	SUMO modification of proteins (sumoylation) is essential for mitotic progression from yeast to humans, but only a limited number of sumoylated proteins with functions in mitosis have been discovered. Vertebrates express three SUMO paralogs, SUMO-1, SUMO-2 and SUMO-3, but only SUMO-2 and -3 are chromosome-associated in mitosis. In this study, we used chromosome spreads to more precisely define the localization of endogenous SUMO-2 and -3 to the centromere as well as the chromosome protein scaffold. Furthermore, we developed methodologies for the immunopurification of endogenous SUMO-2 and -3 modified proteins from cell extracts. Using LC-MS/MS, we analyzed proteins immunopurified from mitotic chromosome fractions and G0 nuclear fractions. We identified 296 putative sumoylated proteins, with 138 being modified specifically in mitosis. We identified proteins known to localize to the centromere and kinetochore and the chromosome protein scaffold, consistent with SUMO-2/3 localization. Furthermore, we demonstrate that utilizing cell synchronization and fractionation allowed for the discovery of low abundance sumoylated proteins that are not detected in large asynchronous analyses. Our results provide a foundation for further characterization of the roles of sumoylation in regulating diverse aspects of chromosome segregation in mitosis. Thermo .raw files were uploaded to the ProHits (Liu et al, 2010) analytical suite and converted to .mzXML format using ReAdW software. Data were searched using X!Tandem (Craig & Beavis, 2004) against human ORFs (RefSeq v45). Search parameters specified a parent MS tolerance of +/- 15ppm, and an MS/MS tolerance of 0.4 Da, with up to two missed cleavages for trypsin. Oxidation of methionine and tryptophan, ubiquitylation of lysine, and alkylation of cysteine (by NEM) were allowed as variable modifications. Statistical validation of the results was performed using Peptide Prophet and Protein Prophet (Keller et al, 2002; Nesvizhskii et al, 2003) as part of the trans-proteomic pipeline. For each search, the Protein Prophet probability at a 1% false discovery rate was used as a cutoff value to generate SAINT-compatible input files. SAINT parameters were as follows: 5000 iterations, low mode Off (0), minFold 1 and normalization (1) (Choi et al, 2011). SAINT cutoff values of 0.75 were used to generate a high-confidence list of protein identifications.
HostingRepository	PRIDE
AnnounceDate	2019-11-27
AnnouncementXML	Submission_2019-11-27_06:54:05.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	tharan srikumar
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	dihydroxylated residue; dehydrated residue; N-ethylmaleimide derivatized cysteine; ubiquitination signature dipeptidyl lysine; monohydroxylated residue; deamidated residue
Instrument	LTQ Orbitrap Velos

Revision	Datetime	Status	ChangeLog Entry
0	2013-07-30 01:29:56	ID requested
1	2019-11-25 02:58:30	announced
⏵ 2	2019-11-27 06:54:06	announced	2019-11-27: Updated project metadata.

Cube, ñ, as-Potts C, Srikumar T, Lee C, Osula O, Subramonian D, Zhang XD, Cotter RJ, Raught B, Matunis MJ, Identification of SUMO-2/3-modified proteins associated with mitotic chromosomes. Proteomics, 15(4):763-72(2015) [pubmed]

curator keyword: Biological

submitter keyword: Human, AP-MS, LC-MS/MS, SUMO, GO, mitosis

Michael J. Matunis
contact affiliation	Department of Biochemistry & Molecular Biology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA
contact email	mmatuni1@jhu.edu
lab head
tharan srikumar
contact affiliation	Medical Biophysics
contact email	tharan.srikumar@gmail.com
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD000381
     1. Label: PRIDE project
     2. Name: Identification of SUMO-2/3-modified proteins associated with mitotic chromosomes