Protein interactions with glycolipids are implicated in diverse cellular processes. However, the study of protein-glycolipid complexes remains a significant experimental challenge. Here, we describe a powerful new assay that combines electrospray ionization mass spectrometry (ESI-MS) and picodiscs, which are composed of human sphingolipid activator protein saposin A and a small number of phospholipids, to display glycolipids in a lipid environment for protein-glycolipid interaction studies in aqueous solution. Time-resolved measurements of enzyme catalyzed hydrolysis of glycolipid substrates and the detection of low, moderate, and high affinity protein-glycolipid interactions serve to demonstrate the reliability and versatility of the assay.

Recognition of terminal sialic acids is central to many cellular processes, and structural modification of sialic acid can disrupt these interactions. A prominent, naturally occurring, modification of sialic acid is 9- O-acetylation (9- O-Ac). Study of this modification through generation and analysis of 9- O-Ac sialosides is challenging because of the lability of the acetate group. Fundamental questions regarding the role of 9- O-Ac sialic acids remain unanswered, including what effect it may have on recognition and hydrolysis by the human neuraminidase enzymes (hNEU). To investigate the substrate activity of 9- O-acetylated sialic acids (Neu5,9Ac), we synthesized an acetylated fluorogenic hNEU substrate 2'-(4-methylumbelliferyl)-9- O-acetyl-α-d- N-acetylneuraminic acid. Additionally, we generated a panel of octyl sialyllactosides containing modified sialic acids including variation in linkage, 9- O-acetylation, and C-5 group (Neu5Gc). Relative rates of substrate cleavage by hNEU were determined using fluorescence spectroscopy and electrospray ionization mass spectrometry. We report that 9- O-acetylation had a significant, and differential, impact on sialic acid hydrolysis by hNEU with general substrate tolerance following the trend of Neu5Ac > Neu5Gc ≫ Neu5,9Ac for NEU2, NEU3, and NEU4. Both NEU2 and NEU3 had remarkably reduced activity for Neu5,9Ac containing substrates. Other isoenzymes appeared to be more tolerant, with NEU4 even showing increased activity on Neu5,9Ac substrates with an aryl aglycone. The impact of these minor structural changes to sialic acid on hNEU activity was unexpected, and these results provide evidence of the substantial influence of 9- O-Ac modifications on hNEU enzyme substrate specificity. Furthermore, these findings may implicate hNEU in processes governed by 9- O-acetyltransferases and -esterases.

Abstract. Tubulin, which is the building block of microtubules, plays an important role in cell division. This critical role makes tubulin an attractive target for the development of chemotherapeutic drugs to treat cancer. Currently, there is no general binding assay for tubulin-drug interactions. The present work describes the application of the catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS) assay to investigate the binding of colchicinoid drugs to αβ-tubulin dimers extracted from porcine brain. Proof-of-concept experiments using positive (ligands with known affinities) and negative (non-binders) controls were performed to establish the reliability of the assay. The assay was then used to screen a library of seven colchicinoid analogues to test their binding to tubulin and to rank their affinities.

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