Synthesis of CID-cleavable protein crosslinking agents containing quaternary amines for structural mass spectrometry

Abstract

Two novel cyclic quaternary amine crosslinking probes are synthesized for structural mass spectrometry of protein complexes in solution and for analysis of protein interactions in organellar and whole cell extracts. Each exhibits high aqueous solubility, excellent protein crosslinking efficiencies, low collision induced dissociation (CID) energy fragmentation efficiencies, high stoichiometries of reaction, increased charges of crosslinked peptide ions, and maintenance of overall surface charge balance of crosslinked proteins.

Cross-linking/mass spectrometry at the crossroads

“…Derived from DSSO, two trifunctional cross-linkers were designed harboring azide (azide-A-DSBSO) or alkyne (alkyne-A-DSBSO) groups to enable affinity purification strategies based on click chemistry [ 20 ]. Similarly, the trifunctional PAC4 linker was developed from DC4 containing an alkyne group for an affinity enrichment of cross-links [ 22 ]. An exciting and novel approach for the enrichment of cross-linked products is provided by the PhoX linker [ 25 ].…”

“…3 Reduction of search space for MS-cleavable cross-linkers. After MS selection of a crosslinked product as a precursor ion, the masses of the connected peptides can be inferred from the fragment ions created in MS/MS experiments trifunctional PAC4 linker was developed from DC4 containing an alkyne group for an affinity enrichment of cross-links [22]. An exciting and novel approach for the enrichment of cross-linked products is provided by the PhoX linker [25].…”

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

“…All currently available MS-cleavable reagents contain labile bonds as cleavage sites in their spacer chains, which are usually fragmented by CID-MS/MS (Table ). ,,,,− One cross-linking reagent has been developed that harbors both CID and ETD cleavability . Also, radical-based MS-cleavable cross-linkers have proven to be useful for FRIPS (free-radical-induced peptide sequencing), as they induce ETD-like fragmentation of peptides .…”

Interfaces with Structure Dynamics of the Workhorses from Cells Revealed through Cross-Linking Mass Spectrometry (CLMS)

“…Representation of the suggested fragmentation schemes (interlink as well as intralink cross-linked peptides) of DSS and DSSO cross-linked peptides. ( d ) In addition to DSS and DSSO, most commonly used cross-linkers are shown (SuDP, Disuccinimidylsuccinamyl aspartyl proline; DSBU, Disuccinimidyldibutyric urea; BS3, Bis(sulfosuccinimidyl) suberate; DC4, 1,4-Bis{4-[(2,5-dioxo-1-pyrrolidinyl) oxy]-4-oxobutyl}-1,4-diazoniabicyclo[2.2.2]octaneoctane; PAC4, 1,1-Bis{4-[(2,5-dioxopyrrolidin-1-yl)oxy]-4-oxobutyl}-4-ethynylpiperidin-1-ium; EDC, 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide; Azide-A-DSBSO, Azide-tagged, acid-cleavable disuccinimidyl bissulfoxide; SDA, succinimidyl 4,4’-azipentanoate; CBDPS, Cyanurbiotindipropionyl succinimide; and PIR, protein interaction reporter) [ 71 , 72 , 89 , 91 , 92 , 93 , 94 , 95 , 96 , 97 , 98 , 99 , 100 ]. ( e ) For the enrichment of cross-linked products/peptides a few specific cross-linkers areused such as:PhoX linker (non-cleavable but containing a phosphonic group) [ 101 ], CLIP cross-linker (introduces alkyne groups to cross-linked peptides) [ 102 ], and 13 C labeled form of biphenyldiglyoxal (diglyoxal cross-linkers) [ 103 , 104 ].…”

“…As derivatives of DSSO, two different trifunctional cross-linkers were designed harboring azide (azide-A-DSBSO: azide-tagged, acid-cleavable disuccinimidyl bissulfoxide) or alkyne (alkyne-A-DSBSO) groups, which enable affinity purification approaches that are based on click-chemistry [ 99 ]. Likewise, the trifunctional PAC4 (1,1-Bis{4-[(2,5-dioxopyrrolidin-1-yl) oxy]-4-oxobutyl}-4-ethynylpiperidin-1-ium) linker was designed from DC4 having an alkyne group for an affinity enrichment of cross-links ( Figure 4 ) [ 71 , 100 ]. The impressive array of chemistry applied for the enrichment of cross-linked products is provided by the PhoX linker ( Figure 4 e) [ 101 ].…”