A synthetic peptide library for benchmarking crosslinking-mass spectrometry search engines for proteins and protein complexes

Rebecca Beveridge,Karl Mechtler,Josef M Penninger,Johannes Stadlmann

doi:10.1038/s41467-020-14608-2

Abstract

Crosslinking-mass spectrometry (XL-MS) serves to identify interaction sites between proteins. Numerous search engines for crosslink identification exist, but lack of ground truth samples containing known crosslinks has precluded their systematic validation. Here we report on XL-MS data arising from measuring synthetic peptide libraries that provide the unique benefit of knowing which identified crosslinks are true and which are false. The data are analysed with the most frequently used search engines and the results filtered to an estimated false discovery rate of 5%. We find that the actual false crosslink identification rates range from 2.4 to 32%, depending on the analysis strategy employed. Furthermore, the use of MS-cleavable crosslinkers does not reduce the false discovery rate compared to non-cleavable crosslinkers. We anticipate that the datasets acquired during this research will further drive optimisation and development of XL-MS search engines, thereby advancing our understanding of vital biological interactions.

Highlights

Crosslinking-mass spectrometry (XL-MS) serves to identify interaction sites between proteins
The crosslinked peptide library was based on the amino acid sequences of tryptic peptides from S. pyogenes Cas[9] and was chemically synthesised according to Fig. 1
To prevent peptide N-termini and Cterminal lysine residues from crosslinking during formation of the library, any C-terminal lysine residues were incorporated into the peptide as epsilon-azido-L-lysine, and the N-terminus of the peptide was protected from crosslinking with a biotin group covalently linked to a generic ‘linker’ peptide sequence YGGGGR, followed by the library peptide sequence (Fig. 1a)

Summary

Introduction

Crosslinking-mass spectrometry (XL-MS) serves to identify interaction sites between proteins. A reagent forms covalent bonds between specific amino acid side-chains that are in close spatial proximity, revealing distance restraints between residues, and interaction sites within a protein or between different proteins[3,4]. Initial applications of these techniques were limited to small proteins and protein complexes. Cleavable crosslinkers generate diagnostic ion doublets during MS2, which are required for the use of novel database search engines such as XlinkX and MeroX that provide means for proteome-wide XL-MS studies[12,13]

Methods

Results

Conclusion