Alkynyl-Enrichable Carboxyl-Selective Crosslinkers to Increase the Crosslinking Coverage for Deciphering Protein Structures

The coverage of chemical crosslinking coupled with mass
spectrometry
(CXMS) is of great importance to determine its ability for deciphering
protein structures. At present, N-hydroxysuccinimidyl
(NHS) ester-based crosslinkers targeting lysines have been predominantly
used in CXMS. However, they are not always effective for some proteins
with few lysines. Other amino acid residues such as carboxyl could
be crosslinked to complement lysines and improve the crosslinking
coverage of CXMS, but the low intrinsic chemical reactivity of carboxyl
compromises the application of carboxyl-selective crosslinkers for
complex samples. To enhance the crosslinking efficiency targeting
acidic residues and realize in-depth crosslinking analysis of complex
samples, we developed three new alkynyl-enrichable carboxyl-selective
crosslinkers with different reactive groups such as hydrazide, amino,
and aminooxy. The crosslinking efficiencies of the three crosslinkers
were systematically evaluated, giving the best reactivity of the amino-functionalized
crosslinker BAP. Furthermore, BAP was extended to the crosslinking
analysis of Escherichia coli lysate
in combination with efficient crosslink enrichment. A total of 1291
D/E–D/E crosslinks involved in 392 proteins were identified
under a false discovery rate (FDR) of ≤1%. Obvious structural
complementarity of BAP was exhibited to the lysine-targeting crosslinker,
facilitating the capability of CXMS for protein structure elucidation.
To the best of our knowledge, this was the first time for the carboxyl-selective
crosslinker to achieve proteome-wide crosslinking analysis of the
whole cell lysate. Collectively, we believe that this work not only
expands on a promising toolkit of CXMS targeting acidic residues but
also provides a valuable guideline to advance the performance of carboxyl-selective
crosslinkers.