An Optimized Miniaturized Filter Aided Sample Preparation Method for Sensitive Cross-linking Mass Spectrometry Analysis of Microscale Samples

ABSTRACT: 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.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Yang Li  

LAB HEAD: Zhenbin, Zhang

PROVIDER: PXD051322 | Pride | 2025-01-03

REPOSITORIES: pride