Mass Spectrometry Compatible Crosslinkers

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 TiO₂ 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).