PXD040010

PXD040010 is an original dataset announced via ProteomeXchange.

Title	Development of a simple and high-efficient method of sample preparation for liver microproteomics analysis
Description	Background and Aims: Liver proteomics with limited sample amounts is technically challenging but valuable for innovative hepatology research. We aimed to establish a simple and high-efficient approach for protein extraction and preparation from the formaldehyde-fixed paraffin-embedded (FFPE) liver samples for microproteomics assessment. Methods: Individual cell collected by laser capture microdissection (LCM) from FFPE liver slices were used as test samples with microscale samples of fresh-frozen liver or cultured liver cells as controls. Protocol for extracting protein was determined through the orthogonal test of 6 conditions of buffer compositions and heating processes. For sample preparation, we adjusted the protocol of single-tube solid-phase sample preparation (SP3) by enhancing protein precipitation and elution and developed a new method named HDMSP. HDMSP was then tested for its capability, reproducibility, and sample-type compatibility in analysis with nanoscale liquid chromatography-tandem mass spectrometry (nano LC-MS/MS). Results: For FFPE protein extraction, adding 4% SDS significantly increased the production by 2.5 times; incubation for 2 hours at 95℃ in alkaline amine buffer improved both the efficiency and quality of protein extraction. For the development of HDMSP, using 2% SDS to elute protein, using 70% acetonitrile but not ethanol, and increasing input of carboxyl magnetic beads to 2 mg/ml improved the rate of protein recovery by 88%, 50%, and 74%, respectively. For either 20 nL FFPE liver or HepG2 cell samples, or 1~2 μg fresh-frozen liver samples, the rate of protein recovery was stable at around 75%. LC-MS/MS demonstrated that the protocol designed for FFPE samples protein extraction allowed for unbiased extraction of insoluble or hydrophobic proteins and with HDMSP, the depth, identification, physicochemical properties, subcellular locations, and reproducibility of FFPE liver microproteome was comparable to those of fresh-frozen sample control. HDMSP also showed high efficiency and reproducibility for subcellular nuclear and cytoplasm-isolated proteins. Conclusion: The new approach including 300mM Tris, 4% SDS incubation for 2h at 95°C, and then preparation with HDMSP is simple, robust, and highly efficient for use in microproteomics of FFPE liver samples. This protocol can provide a solution reference for liver FFPE microproteomics.
HostingRepository	iProX
AnnounceDate	2023-04-25
AnnouncementXML	Submission_2023-04-25_01:26:04.865.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Yonger Wang
SpeciesList	scientific name: Mus musculus; NCBI TaxID: 10090;
ModificationList	No PTMs are included in the dataset
Instrument	Orbitrap Fusion

Revision	Datetime	Status	ChangeLog Entry
0	2023-02-09 19:41:17	ID requested
⏵ 1	2023-04-25 01:26:05	announced

Dataset with its publication pending

submitter keyword: Microproteomics, formaldehyde-fixed paraffin-embedded tissue,protein extraction,detergent compatibility,sample preparation,liver

Yan Wang
contact affiliation	Biomedical Research Center, Southern Medical University, Guangzhou, China
contact email	yanwang@smu.edu.cn
lab head
Yonger Wang
contact affiliation	Biomedical Research Center, Southern Medical University
contact email	yongerwang010763@163.com
dataset submitter

iProX dataset URI