PXD051322-1

PXD051322 is an original dataset announced via ProteomeXchange.

Title	An Optimized Miniaturized Filter Aided Sample Preparation Method for Sensitive Cross-linking Mass Spectrometry Analysis of Microscale Samples
Description	Cross-linking mass spectrometry (XL-MS) is a powerful tool for elucidating protein structures and protein-protein interactions (PPIs) at the global scale. However, sensitive XL-MS analysis of mass-limited samples remains challenging, due to serious sample loss during sample preparation of the low-abundance cross-linked peptides. Herein, an optimized miniaturized filter aided sample preparation (O-MICROFASP) method was presented for sensitive XL-MS analysis of microscale samples. By systematically investigating and optimizing crucial experimental factors, this approach dramatically improves the XL identification of low and sub-microgram samples. Compared with conventional FASP method, more than 7.4 times cross-linked peptides were identified from single-shot analysis of 1 µg DSS cross-linked HeLa cell lysates (440 vs 59). The number of cross-linked peptides identified from 0.5 µg HeLa cell lysates was increased by 58% when further reducing the surface area of the filter to 0.058 mm2 in the microreactor. To deepen the identification coverage of XL-proteome, five different types of cross-linkers were used and each µg of cross-linked HeLa cell lysates was processed by O-MICROFASP integrated with tip-based strong cation exchange (SCX) fractionation. Up to 2741 unique cross-linked peptides were identified from the 5 µg HeLa cell lysates, representing 2579 unique K-K linkages on 1092 proteins. ~96% of intraprotein cross-links were within the maximal distance restraints of 26 Å, and 75% of the identified PPIs reported by STRING database were with high confidence (scores ≥ 0.9), confirming the high validity of the identified cross-links for protein structural mapping and PPI analysis. This study demonstrates that O-MICROFASP is a universal and efficient method for proteome-wide XL-MS analysis of microscale samples with high sensitivity and reliability.
HostingRepository	PRIDE
AnnounceDate	2025-01-03
AnnouncementXML	Submission_2025-01-03_04:30:47.801.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Yang Li
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	No PTMs are included in the dataset
Instrument	Orbitrap Fusion Lumos

Revision	Datetime	Status	ChangeLog Entry
0	2024-04-09 20:47:38	ID requested
⏵ 1	2025-01-03 04:30:48	announced

10.1021/ACS.ANALCHEM.4C01600;

submitter keyword: Cross-linking mass spectrometry,Microscale samples

Zhenbin, Zhang
contact affiliation	Institute of Drug Discovery Technology, Ningbo University
contact email	zhangzhenbin@nbu.edu.cn
lab head
Yang Li
contact affiliation	Ningbo University
contact email	liyang@nbu.edu.cn
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD051322
     1. Label: PRIDE project
     2. Name: An Optimized Miniaturized Filter Aided Sample Preparation Method for Sensitive Cross-linking Mass Spectrometry Analysis of Microscale Samples