Characterization of the Zebrafish Homolog of ß-Glucosidase 2: A Target of the Drug Miglustat.

The small-molecular compound miglustat (N-butyldeoxynojirimycin, Zavesca(®)) has been approved for clinical use in type 1 Gaucher disease and Niemann-Pick type C disease, which are disorders caused by dysfunction of the endosomal-autophagic-lysosomal system. Miglustat inhibits a number of enzymes involved in glycoconjugate and glycan metabolism, including ß-glucosidase 2 (GBA2), which is exceptionally sensitive to inhibition by miglustat. GBA2 is a glucosylceramide-degrading enzyme that is located on the plasma membrane/endoplasmic reticulum, and is distinct from the lysosomal enzyme glucocerebrosidase (GBA). Various strands of evidence suggest that inhibition of GBA2 contributes to the therapeutic benefits of miglustat. To further explore the pharmacology and biology of GBA2, we investigated whether the zebrafish homolog of GBA2 has similar enzymatic properties and pharmacological sensitivities to its human counterpart. We established that zebrafish has endogenous ß-glucosidase activity toward lipid- and water-soluble GBA2 substrates, which can be inhibited by miglustat, N-butyldeoxygalactonojirimycin, and conduritol B epoxide. ß-Glucosidase activities with highly similar characteristics were expressed in cells transfected with the zebrafish gba2 cDNA and in cells transfected with the human GBA2 cDNA. These results provide a foundation for the use of zebrafish in screening GBA2-targeting molecules, and for wider studies investigating GBA2 biology.

Verification and refinement of cellular glycosphingolipid profiles using HPLC.

Glycosphingolipids (GSLs) are hybrid molecules consisting of the sphingolipid ceramide linked to a mono- or oligo-saccharide. In comparison to other membrane lipids, the family of GSLs stands out because of the extensive variation in the carbohydrate headgroup. GSLs are cell surface binding partners, in cis with growth factor receptors, and in trans with bacterial toxins and viruses, and are among the host-derived membrane components of viral particles, including those of HIV. In spite of their biological relevance, GSL profiles of commonly used cell lines have been analyzed to different degrees. Here, we directly compare the GSL complements from CHO-K1, COS-7, HeLa, HEK-293, HEPG2, Jurkat, and SH-SY5Y cells using an HPLC-based method requiring modest amounts of material. Compared to previous studies, the HPLC-based analyses provided more detailed information on the complexity of the cellular GSL complement, qualitatively as well as quantitatively. In particular for cells expressing multiple GSLs, we found higher numbers of GSL species, and different levels of abundance. Our study thus extends our knowledge of biologically relevant lipids in widely used cell lines.

Resolubilization of precipitated intact membrane proteins with cold formic acid for analysis by mass spectrometry.

Protein precipitation in organic solvent is an effective strategy to deplete sodium dodecyl sulfate (SDS) ahead of MS analysis. Here we evaluate the recovery of membrane and water-soluble proteins through precipitation with chloroform/methanol/water or with acetone (80%). With each solvent system, membrane protein recovery was greater than 90%, which was generally higher than that of cytosolic proteins. With few exceptions, residual supernatant proteins detected by MS were also detected in the precipitation pellet, having higher MS signal intensity in the pellet fraction. Following precipitation, we present a novel strategy for the quantitative resolubilization of proteins in an MS-compatible solvent system. The pellet is incubated at -20 °C in 80% formic acid/water and then diluted 10-fold with water. Membrane protein recovery matches that of sonication of the pellet in 1% SDS. The resolubilized proteins are stable at room temperature, with no observed formylation as is typical of proteins suspended in formic acid at room temperature. The protocol is applied to the molecular weight determination of membrane proteins from a GELFrEE-fractionated sample of Escherichia coli proteins.

Perfluorooctanoic acid and ammonium perfluorooctanoate: volatile surfactants for proteome analysis?

RATIONALE: Fluorinated surfactants are being explored as mass spectrometry (MS)-friendly alternatives to sodium dodecyl sulfate (SDS) for proteome analysis. Previous work demonstrates perfluorooctanoic acid (PFOA) to be compatible with electrospray ionization (ESI)-MS. The high volatility of PFOA provides an intrinsic approach to potentially eliminate the surfactant during ESI, or alternatively through solvent evaporation prior to MS. The ammonium salt of PFOA, ammonium perfluorooctanoate (APFO), is likely favored for proteome experiments; the MS and liquid chromatography (LC)/MS tolerance of APFO has not been established for proteome applications. METHODS: Standard proteins and peptides, as well as a yeast proteome mixture, were individually spiked with surfactants (APFO, PFOA, SDS), and subjected to direct infusion ESI-MS, LC/MS/MS and LC/UV. The level of fluorinated surfactant remaining after solvent evaporation under varying conditions (time, pH, salt and protein content) was quantified and compared to the threshold tolerance level of the surfactant in an MS experiment (determined herein). RESULTS: Whereas PFOA is found ineffective at assisting protein solubilization, APFO is as effective as SDS for resolubilization of acetone-precipitated yeast proteins (~100% recovery). Unfortunately, the LC and MS threshold tolerance of APFO is only minimally greater than SDS (~2-fold higher concentration to cause 50% suppression). Nonetheless, the benefits of APFO in a proteome experiment are realized following a one-step evaporation protocol for removal of the surfactant in acidified solvent. CONCLUSIONS: APFO is considered a favoured alternative to SDS for proteome solubilization. Strictly speaking, APFO is not an 'MS-friendly' surfactant for proteome characterization; the detergent not only suppresses ESI signals at high concentration, but also perturbs reversed phase separation. However, the simplicity of APFO removal ahead of LC/MS justifies its use over the conventional SDS.

Top-down and bottom-up proteomics of SDS-containing solutions following mass-based separation.

SDS has recognized benefits for protein sample preparation, including solubilization and imparting molecular weight separation (e.g., SDS-PAGE). Here, we compare two proteome workflows which incorporate SDS for protein separation, namely, SDS-PAGE coupled to LC/MS (GeLC MS), along with a solution separation platform, GELFrEE, for intact proteome prefractionation and identification. Despite the clear importance of SDS in these and other proteome analysis workflows, the affect of SDS on an LC/MS proteome experiment has not been quantified. We first examined the influence of SDS on both a bottom-up as well as a top-down (intact protein) MS workflow. Surprisingly, at levels up to 0.01% SDS in the injected sample, reliable MS characterization is obtained. We subsequently explored organic precipitation protocols (chloroform/methanol/water and acetone) as a means of lowering SDS, and present a simple modified acetone precipitation protocol which consistently enables MS proteome characterizations from samples initially containing 2% SDS. With this effective strategy for SDS reduction, the GELFrEE MS workflow for bottom-up proteome analysis was characterized relative to GeLC MS. Remarkable agreement in the number and type of identified proteins was obtained from these two separation platforms, validating the use of SDS in solution-phase proteome analysis.