PXD018248

PXD018248 is an original dataset announced via ProteomeXchange.

Title	Towards a universal sample preparation method for denaturing top-down proteomics of complex proteomes
Description	A universal and standardized sample preparation method becomes vital for denaturing top-down proteomics (dTDP) to advance the scale and accuracy of proteoform delineation in complex biological systems. It needs to have high protein recovery, minimum bias, good reproducibility, and compatibility with downstream mass spectrometry (MS) analysis. Here we employed a lysis buffer containing sodium dodecyl sulfate (SDS) for extracting proteoforms from cells, and for the first time, compared membrane ultrafiltration (MU), chloroform-methanol precipitation (CMP), and single-spot solid-phase sample preparation using magnetic beads (SP3) for proteoform cleanup for dTDP. The MU method outperformed CMP and SP3 methods, resulting in high and reproducible protein recovery from both E. coli cell (59±3%) and human HepG2 cell (86±5%) samples without a significant bias. Single-shot capillary zone electrophoresis (CZE)-MS/MS analyses of the prepared E. coli and HepG2 cell samples using the MU method identified 821 and 516 proteoforms, respectively. Nearly 30% and 50% of the identified E. coli and HepG2 proteins are membrane proteins. CZE-MS/MS identified 94 histone proteoforms from the HepG2 sample with various post-translational modifications, including acetylation, methylation, and phosphorylation. Our results suggest that combining the SDS-based protein extraction and the MU-based protein cleanup could be a universal sample preparation method for dTDP.
HostingRepository	PRIDE
AnnounceDate	2020-05-22
AnnouncementXML	Submission_2020-05-21_18:59:57.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Liangliang Sun
SpeciesList	scientific name: Escherichia coli; NCBI TaxID: 562; scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	monomethylated residue; phosphorylated residue; acetylated residue
Instrument	Q Exactive

Revision	Datetime	Status	ChangeLog Entry
0	2020-03-30 00:22:36	ID requested
⏵ 1	2020-05-21 18:59:58	announced

Yang Z, Shen X, Chen D, Sun L, Toward a Universal Sample Preparation Method for Denaturing Top-Down Proteomics of Complex Proteomes. J Proteome Res, 19(8):3315-3325(2020) [pubmed]

submitter keyword: sample preparation, denaturing top-down proteomics, SDS, membrane ultrafiltration, chloroform-methanol precipitation, SP3, CZE-MS/MS, membrane proteins, histone, PTMs

Liangliang Sun
contact affiliation	Department of Chemistry, Michigan State University
contact email	lsun@chemistry.msu.edu
lab head
Liangliang Sun
contact affiliation	Michigan State University
contact email	lsun@chemistry.msu.edu
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD018248
     1. Label: PRIDE project
     2. Name: Towards a universal sample preparation method for denaturing top-down proteomics of complex proteomes