Thermo Scientific™ Mass Spectrometry Cleavable Crosslinkers

Description

• Chemical crosslinking in combination with mass spectrometry is a powerful method to improve protein characterization and identification of protein-protein interactions
• This method has been applied to recombinant and native protein complexes and, more recently, to whole cell lysates or intact unicellular organisms in efforts to identify protein-protein interactions on a global scale
• The more recent enhancement of cleavability in DSSO and DSBU has improved MS compatibility and sensitivity of detection of crosslinked peptides and proteins
• DSSO (disuccinimidyl sulfoxide) and DSBU (disuccinimidyl dibutyric urea or BuUrBU) are both mass spectrometry (MS)-cleavable and membrane-semi-permeable crosslinkers that contain an amine-reactive N-hydroxysuccinimide (NHS) ester at each end of a 7-carbon spacer arm (DSSO) or 11-atom spacer arm (DSBU)
• Both crosslinkers have reactivity similar to DSS but contain a cleavable functionality in the linker region that enables cleavage in the gas phase using collision-induced dissociation (CID) during tandem MS (MS/MS) fragmentation
• The MS cleavage of DSSO or DSBU generates diagnostic ion doublets during MS2, which distinguishes identification of crosslinked peptides from dead-end modifications
• The crosslinked peptide fragments are subsequently identified using MeroX or XlinkX software
• For DSBU, crosslinked peptides are fragmented and sequenced during MS2, while for DSSO, crosslinked peptides are fragmented during MS/MS and require additional fragmentation (MS3) to facilitate peptide sequencing using traditional database search engines
• Features of MS-cleavable crosslinkers DSSO and DSBU • Highly reactive NHS ester (at both ends) with primary amines • MS-cleavable by collision-induced dissociation (CID) • High-purity crystalline reagents for protein structure and interaction characterization • Membrane-semi-permeable, allowing intracellular crosslinking • Water-insoluble (dissolve first in DMF or DMSO) Benefits of MS-cleavable crosslinkers Although both non-cleavable and MS-cleavable crosslinkers can be used to study protein-protein interactions, MS-cleavable crosslinkers may be more advantageous to improve identification and analysis of crosslinked peptides
• Typically when using non-cleavable crosslinkers, the data generated consists of both the crosslinked and non-crosslinked peptides, making it difficult to distinguish using mass spectrometry analysis
• Non-cleavable crosslinkers also increase the complexity of the analysis
• Some complexity can be reduced through additional sample preparation or through isotopic labeling
• MS-cleavable crosslinkers provide the ability to distinguish between crosslinked and non-crosslinked peptides for analysis
• The crosslinker is cleaved through collision induced dissociation (CID/HCD) or electron transfer induced dissociation (ETD) to generate characteristic ions that can then be used during MS2 or MS2/MS3 fragmentation to confidently identify and sequence crosslinked peptides
• The ability to unambiguously identify crosslinked peptides results in fewer false positives when compared to non-cleavable crosslinkers
• DSSO and DSBU utilize a sulfoxide or urea, respectively, for cleavability with gas phase fragmentation
• Applications for MS-cleavable crosslinkers DSSO and DSBU • Analyzing complex protein mixtures • Profiling native protein-protein networks in vivo • Investigating intact organelles, cells, or tissues • Performing proteome-wide studies • Characterizing intermolecular protein interactions • Characterizing purified proteins and complexes Note: For high resolution analysis of the crosslinked peptides, the recommended LC column for the Nanospray Flex source is the Acclaim PepMap 100 C18 LC Column (Cat
• No
• 164940 or 164568)
• For the EASY-Spray source, the recommended LC column is the EASY-Spray C18 LC Column (Cat
• No
• ES900 or ES904).