Thermo Scientific™ Mass Spectrometry Compatible Crosslinkers

Description

• Chemical crosslinking in combination with mass spectrometry is a powerful method to improve protein characterization, and to identify 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 enrichment feature of DSPP and TBDSPP improves detection of crosslinked proteins by reducing sample complexity through separation of crosslinked from non-crosslinked peptides
• • Tri-functional crosslinker: NHS ester (both ends), phosphonic acid (DSPP), or phosphonate ester (TBDSPP) in the spacer for enrichment • High-purity crystalline reagents for protein structure and interaction characterization • Membrane-permeable version (TBDSPP) for intracellular crosslinking • Enrichable using Fe-NTA IMAC or TiO 2 MOAC DSPP (disuccinimidyl phenyl phosphonic acid, PhoX) and TBDSPP (tert-butyl disuccinimidyl phenyl phosphonate, tBu-PhoX) are phospho-enrichable crosslinkers containing an amine-reactive N-hydroxysuccinimide (NHS) ester at each end of a 7-atom spacer arm with a phosphonic acid group (DSPP) or phosphonate ester (TBDSPP) for enrichment
• Both react efficiently with primary amine groups (-NH2) in pH 7–9 buffers to form stable amide bonds
• The phosphonic acid or phosphonate ester groups can then be used to enrich crosslinked peptides using immobilized metal affinity chromatography (IMAC) or metal oxide affinity chromatography (MOAC)
• Of the two enrichable MS crosslinkers, DSPP is more water soluble than TBDSPP and is better for in vitro crosslinking, simple purified proteins and protein complexes
• TBDSPP is better for intracellular crosslinking due to increased membrane permeability
• Although both crosslinkers can be enriched using traditional phosphopeptide methods, TBDSPP crosslinked peptides must first be incubated with TFA to remove the tert-butyl protection groups
• The TBDSPP phosphonic acid group (after acid cleavage) is not a substrate for phosphatases, which provides opportunity for the removal endogenous phosphopeptides in samples prior to enrichment
• 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).