PXD037491

PXD037491 is an original dataset announced via ProteomeXchange.

Title	Optimization of Higher-Energy Collisional Dissociation Fragmentation Energies for Intact Protein-level Tandem Mass Tag Labeling
Description	Isobaric chemical tag labeling (e.g., iTRAQ and TMT) is a commonly used approach in quantitative proteomics research. Typically, peptides are covalently labeled with isobaric chemical tags, and quantification is enabled through detection of low-mass reporter ions generated after MS2 fragmentation. Recently, we have introduced and optimized a platform for intact protein-level TMT labeling that demonstrated >90% labeling efficiency in complex sample with top-down proteomics. Higher-energy collisional dissociation (HCD) is a commonly utilized fragmentation method for peptide-level isobaric chemical tag labeling because it produces accurate reporter ion intensities and avoids the loss of low mass ions. HCD energies have been optimized for peptide-level isobaric chemical tag labeling; however, fragmentation energies have not been systematically evaluated for TMT-labeled intact proteins for both protein identification and quantitation. In this study, we report a systematic evaluation of normalized HCD fragmentation energies on TMT-labeled HeLa lysate with top-down proteomics. Our results suggested that reporter ions often require higher collisional energy for higher ion intensities while most of intact proteins fragment when normalized HCD energies are between 30% and 50%. We further demonstrated that a stepped HCD fragmentation scheme with energies between 30 and 50% resulted in the optimized quantitation and identification for TMT-labeled intact HeLa protein lysate by providing average reporter ion intensity as > 3.60 E4 and average PrSM as > 1000 PrSM counts with high confidence.
HostingRepository	PRIDE
AnnounceDate	2023-11-14
AnnouncementXML	Submission_2023-11-14_07:55:33.502.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Yanting Guo
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	TMT6plex-126 reporter+balance reagent acylated residue
Instrument	Orbitrap Exploris 240

Revision	Datetime	Status	ChangeLog Entry
0	2022-10-17 01:30:48	ID requested
1	2023-01-17 14:45:24	announced
⏵ 2	2023-11-14 07:55:34	announced	2023-11-14: Updated project metadata.

Guo Y, Chowdhury T, Seshadri M, Cupp-Sutton KA, Wang Q, Yu D, Wu S, Optimization of Higher-Energy Collisional Dissociation Fragmentation Energy for Intact Protein-Level Tandem Mass Tag Labeling. J Proteome Res, 22(5):1406-1418(2023) [pubmed]

submitter keyword: protein-level TMT labeling

top-down proteomics

quantitative proteomics

HCD

Si Wu
contact affiliation	University of Oklahoma
contact email	si.wu@ou.edu
lab head
Yanting Guo
contact affiliation	University of Oklahoma
contact email	Yanting.Guo-1@ou.edu
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD037491
     1. Label: PRIDE project
     2. Name: Optimization of Higher-Energy Collisional Dissociation Fragmentation Energies for Intact Protein-level Tandem Mass Tag Labeling