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

Abstract

The coverage of chemical crosslinking coupled with mass spectrometry (CXMS) is of great importance to determine its ability for deciphering protein structures. At present, Nhydroxysuccinimidyl (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 carboxylselective 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.

Chemical reagents for the enrichment of modified peptides in MS-based identification

“…Consideration of the steric hindrance of biotin that may affect the linking efficiency, smaller functionalities, such as alkyne-tagged cross-linkers, have been broadly developed. 32,43,44 Accordingly, azide biotin reagents have been developed allowing for appending biotin through click chemistry after the protein labelling. Additionally, diverse cleavable moieties have been inserted between azide and biotin, producing cleavable reagents for the purpose of releasing peptides.…”

Twisting Urea- to Imide-Based Mass Spectrometry-Cleavable Cross-Linkers Enables Affinity Tagging

“…Also, several cross-linkers incorporate molecular tags like biotin, azide [9], alkyne [9,10], or phosphonate [11], enabling affinity purification of cross-links. The combination of affinity purification followed by SEC or SCX allows reducing the background of linear peptides in the sample [12][13][14]. Among the affinity handles, the phosphonate has recently emerged as one of the more promising options in system-wide XL-MS [12].…”

Evaluating Imide-Based Mass Spectrometry-Cleavable Cross-Linkers for Structural Proteomics Studies

“…Also, several cross-linkers incorporate molecular tags like biotin, azide, alkyne, , or phosphonate, enabling affinity purification of cross-links. The combination of affinity purification followed by SEC or SCX allows reducing the background of linear peptides in the sample. − Among the affinity handles, the phosphonate has recently emerged as one of the more promising options in system-wide XL-MS . Phosphonate-containing cross-links can be enriched by immobilized metal ion affinity chromatography (IMAC) or on titanium dioxide (TiO 2 ) beads.…”