PXD021489

PXD021489 is an original dataset announced via ProteomeXchange.

Title	BAC-DROP: Rapid digestion of proteome fractionated via dissolvable polyacrylamide gel electrophoresis and its application to bottom-up proteomics workflow
Description	The GeLC-MS workflow, which combines low-cost, easy-to-use SDS-polyacrylamide gel electrophoresis (SDS-PAGE) with liquid chromatography-mass spectrometry (LC-MS), is very popular in current bottom-up proteomics. However, GeLC-MS requires that PAGE-separated proteins undergo overnight enzymatic digestion in a gel, resulting in more than 20 hours of sample preparation for LC-MS. In this study, we overcame the limitations of GeLC-MS by developing a rapid digestion workflow for PAGE separation proteins using N,N'-bis(acryloyl)cystamine (BAC) cross-linked gels that can be solubilized by reductive treatment. Making use of an established workflow called BAC-DROP (BAC-gel dissolution to digest PAGE-resolved objective proteins), crude proteome samples were fractionated based on molecular weight by BAC cross-linked PAGE. After fractionation, the gel fragments were reductively dissolved in under 5 minutes, and in-solution trypsin digestion of the protein released from the gel was completed in less than 1 hour at 70 °C, equivalent to a 90–95 % reduction in time compared to conventional in-gel trypsin digestion. The introduction of the BAC-DROP workflow to the MS assays for inflammatory biomarker CRP and viral marker HBsAg allowed for serum sample preparation to be completed in as little as 5 hours, demonstrating successful marker quantification from a 0.5 μL sample of human serum. Combining reduced proteome complexity via PAGE fractionation with rapid trypsin digestion provides a unique opportunity for conducting high-throughput analysis of in-depth proteome with a focus on specific molecular weight ranges.
HostingRepository	PanoramaPublic
AnnounceDate	2021-01-24
AnnouncementXML	Submission_2021-01-24_19:42:51.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Supported dataset by repository
PrimarySubmitter	Nobuaki Takemori
SpeciesList	scientific name: Homo sapiens; NCBI TaxID: 9606; scientific name: Bos taurus; NCBI TaxID: 9913; scientific name: Hepatitis B virus; NCBI TaxID: 10407;
ModificationList	Label:13C(6)15N(2); Label:13C(6)15N(4)
Instrument	QTRAP 5500

Revision	Datetime	Status	ChangeLog Entry
0	2020-09-15 05:46:34	ID requested
1	2020-12-25 13:13:20	announced
⏵ 2	2021-01-24 19:42:52	announced	: Added PubMed ID

Takemori A, Ishizaki J, Nakashima K, Shibata T, Kato H, Kodera Y, Suzuki T, Hasegawa H, Takemori N, BAC-DROP: Rapid Digestion of Proteome Fractionated via Dissolvable Polyacrylamide Gel Electrophoresis and Its Application to Bottom-Up Proteomics Workflow. J Proteome Res, 20(3):1535-1543(2021) [pubmed]

submitter keyword: BAC-PAGE, Bottom-up proteomics, Dissolvable polyacrylamide gel, GeLC-MS, Rapid enzymatic digestion

Nobuaki Takemori
contact affiliation	Ehime University
contact email	takemori@m.ehime-u.ac.jp
lab head
Nobuaki Takemori
contact affiliation	Ehime University
contact email	takemori@m.ehime-u.ac.jp
dataset submitter

Panorama Public dataset URI