Expanding the Cross-Link Coverage of a Carboxyl-Group Specific Chemical Cross-Linking Strategy for Structural Proteomics Applications

Abstract

Carboxyl-group specific chemical cross-linking is gaining an increased interest as a structural mass spectrometry/structural proteomics technique that is complementary to the more commonly used amine-specific chemistry using succinimide esters. One of these protocols uses a combination of dihydrazide linkers and the coupling reagent DMTMM [4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium] chloride, which allows performing the reaction at neutral pH. The reaction yields two types of products, carboxyl–carboxyl cross-links that incorporate the dihydrazide linker and zero-length carboxyl-amine cross-links induced by DMTMM alone. Until now, it has not been systematically investigated how the balance between the two products is affected by experimental conditions. Here, we studied the role of the ratios of the two reagents (using pimelic dihydrazide and DMTMM) and demonstrate that the concentration of the two reagents can be systematically adjusted to favor one reaction product over the other. Using a set of five model proteins, we observed that the number of identified cross-linked peptides could be more than doubled by a combination of three different reaction conditions. We also applied this strategy to the bovine 20S proteasome and the Escherichia coli 70S ribosome, again demonstrating complementarity and increased cross-link coverage.

The human GID complex engages two independent modules for substrate recruitment

“…Two different cross‐linking protocols were used, based on the amine‐reactive disuccinimidyl suberate (DSS) (Leitner et al , 2013) and a combination of pimelic dihydrazide (PDH) and the coupling reagent 4‐(4,6‐dimethoxy‐1,3,5‐triazin‐2‐yl)‐4‐methylmorpholinium (DMTMM) chloride (Leitner et al , 2014; Mohammadi et al , 2021). DSS was obtained as a 1:1 mixture of “light” (d 0 ) and “heavy” (d 12 ) isotopic variants from Creative (d 0 ) PDH from ABCR, heavy (d 10 ) PDH and DMTMM chloride from Sigma‐Aldrich.…”

“…Cross‐linking conditions were optimized in screening experiments on the 5‐subunit hGID complex using SDS–PAGE as a readout, and 1 mM DSS (d 0 /d 12 ) and 22 mM PDH (d 0 /d 10 ) + 4.4 mM DMTMM were selected as the optimal conditions. The low concentration of DMTMM relative to PDH results in the dominant formation of zero‐length cross‐links over the integration of the dihydrazide linker (Mohammadi et al , 2021). For XL‐MS, protein complexes were prepared at a total protein concentration of 1 mg/ml in a buffer containing 50 mM HEPES pH 7.4, 200 mM NaCl, and 1 mM TCEP and cross‐linked at 50 µg scale.…”

Cross-Linking Mass Spectrometry for Investigating Protein Conformations and Protein–Protein Interactions─A Method for All Seasons

“…All these approaches targeting carboxyl groups exhibit several limitations, which require a careful control of reaction conditions in order not to generate artifacts. Very recently, a careful systematic study was performed optimizing the reaction conditions for DMTMM and PDH with respect to the formation of zero-length (carboxyl-amine) cross-links versus hydrazide (carboxyl-carboxyl) cross-links in proteins …”

“…Very recently, a careful systematic study was performed optimizing the reaction conditions for DMTMM and PDH with respect to the formation of zero-length (carboxyl-amine) cross-links versus hydrazide (carboxyl-carboxyl) cross-links in proteins. 88…”

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

“…The initial approach was using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) followed by N -hydroxysuccinimide (NHS) or N -hydroxybenzotriazole (HOBt), requiring a pH of 5.5–6.0 to favor the crosslinking. , As an improvement, the coupling reagent 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) was employed, due to its suitability to bridge the adjacent carboxylate and lysine residues under physiological conditions . To improve the crosslinking coverage and gain more protein structural information, homobifunctional crosslinkers containing amino or hydrazide with a certain crosslinking restraint were developed, such as commercial 1,6-hexanediamine (C6DA) adipic dihydrazide , or pimelic acidic dihydrazide (PDH). , Recently, a hydrazide-based crosslinker, dihydrazide sulfoxide (DHSO), was adopted to complement NHS ester-based DSSO, providing new insights into the structural dynamics of the ninth subunit in the human COP9 signalosome . In addition, a high-density crosslinking strategy was also achieved by coupling the carboxyl-selective crosslinker 2,2′-(ethylenedioxy)­diethylamine with the traditional lysine-targeting crosslinker BS3, demonstrating the high precision for de novo modeling of proteasome regulatory particle architectures .…”