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

Area of Science:

• Proteomics
• Structural Biology
• Chemical Biology

Background:

• Chemical crosslinking coupled with mass spectrometry (CXMS) is crucial for protein structure determination.
• Current CXMS methods predominantly use lysine-targeting crosslinkers, which are limited for proteins with few lysine residues.
• Carboxyl-selective crosslinking offers complementary coverage but faces challenges due to low reactivity.

Purpose of the Study:

• To develop novel alkynyl-enrichable, carboxyl-selective crosslinkers to improve CXMS efficiency for acidic residues.
• To evaluate and identify the most effective crosslinker for complex biological samples.
• To demonstrate the application of the optimized crosslinker for proteome-wide analysis.

Main Methods:

• Synthesis and evaluation of three new carboxyl-selective crosslinkers with varying reactive groups (hydrazide, amino, aminooxy).
• Systematic assessment of crosslinking efficiencies.
• Application of the best-performing crosslinker (BAP) to analyze Escherichia coli lysate using efficient crosslink enrichment.

Main Results:

• The amino-functionalized crosslinker, BAP, exhibited the highest reactivity among the developed compounds.
• Proteome-wide analysis of E. coli lysate using BAP identified 1291 D/E-D/E crosslinks in 392 proteins at ≤1% FDR.
• BAP demonstrated structural complementarity to lysine-targeting crosslinkers, enhancing CXMS capabilities.

Conclusions:

• Developed novel carboxyl-selective crosslinkers, expanding the CXMS toolkit for targeting acidic residues.
• Achieved the first proteome-wide crosslinking analysis of whole cell lysate using carboxyl-selective crosslinkers.
• The study provides valuable insights for advancing carboxyl-selective crosslinker performance in complex proteomic analyses.