A Novel Bio-Orthogonal Cross-Linker for Improved Protein/Protein Interaction Analysis

Abstract

The variety of protein cross-linkers developed in recent years illustrates the current requirement for efficient reagents optimized for mass spectrometry (MS) analysis. To date, the most widely used strategy relies on commercial cross-linkers that bear an isotopically labeled tag and N-hydroxysuccinimid-ester (NHS-ester) moieties. Moreover, an enrichment step using liquid chromatography is usually performed after enzymatic digestion of the cross-linked proteins. Unfortunately, this approach suffers from several limitations. First, it requires large amounts of proteins. Second, NHS-ester cross-linkers are poorly efficient because of their fast hydrolysis in water. Finally, data analysis is complicated because of uneven fragmentation of complex isotopic cross-linked peptide mixtures. We therefore synthesized a new type of trifunctional cross-linker to overrule these limitations. This reagent, named NNP9, comprises a rigid core and bears two activated carbamate moieties and an azido group. NNP9 was used to establish intra- and intermolecular cross-links within creatine kinase, then to map the interaction surfaces between α-Synuclein (α-Syn), the aggregation of which leads to Parkinson's disease, and the molecular chaperone Hsc70. We show that NNP9 cross-linking efficiency is significantly higher than that of NHS-ester commercial cross-linkers. The number of cross-linked peptides identified was increased, and a high quality of MS/MS spectra leading to high sequence coverage was observed. Our data demonstrate the potential of NNP9 for an efficient and straightforward characterization of protein-protein interfaces and illustrate the power of using different cross-linkers to map thoroughly the surface interfaces within protein complexes.

Structural basis for loading and inhibition of a bacterial T6 SS phospholipase effector by the VgrG spike

“…To provide detailed information on the VgrG-Tle1 interaction, the S VgrG-Tle1 H complex was subjected to cross-linking with the NNP9 cross-linker (Nury et al, 2015). NNP9 was specifically engineered to carry 2 NHS carbamate reactive groups, which are less prone to hydrolysis than widely used NHS esters, thus improving cross-linking efficiency and performance compared to the commercial ones (Nury et al, 2015). Mass spectrometry analysis of crosslinked peptides revealed that VgrG and Tle1 establish numerous contacts (Fig 1A,Dataset EV1).…”

Polypeptides derived from α-Synuclein binding partners to prevent α-Synuclein fibrils interaction with and take-up by cells

“…Moreover, the K D were similar to that we determined for full-length Hsc70 (Fig 4B). Thus, both Hsc70 SBDβ and SBD-lid contribute to fibrillar αSyn binding as for monomeric αSyn [37,38].…”

“…Hsc70 is composed of two domains, a Nucleotide Binding Domain (NBD), responsible for the chaperone ATPase activity, and a Substrate Binding Domain (SBD), that binds Hsc70 clients. We previously used lysine-reactive chemical cross-linkers and mass-spectrometry to map the surface areas within Hsc70 that interact with monomeric αSyn; all the identified areas were within the SBD (Fig 3A) [37,38]. To determine whether Hsc70 SBD retains the ability to bind αSyn fibrils we expressed and purified it.…”

“…Incidentally over 60 peptide drugs have now reached the market [ 35 ]. Using a cross-linking and mass spectrometry strategy, we previously mapped the surface interfaces between αSyn monomers or fibrils and two protein partners, namely, the molecular chaperone Hsc70 [ 36 – 38 ] and the sodium/potassium pump Na+/K+-ATPase (NKA) [ 25 ]. Here, we designed a set of polypeptides based on the Hsc70 and NKA surface areas we identified to interact with αSyn.…”

Advances in protein complex analysis by chemical cross-linking coupled with mass spectrometry (CXMS) and bioinformatics

“…The affinity enrichment technique is particularly useful for detection of low abundance cross-linked peptides. Affinity tagging groups [4], such as biotin [34,38,[40][41][42] antibodies, alkyne, phosphate [4], and azido [43], enable selective purification of cross-linked products by chromatography or binding to solid surface.…”