Desorption Electrospray Ionization Mass Spectrometry Assay for Label‐Free Characterization of SULT2B1b Enzyme Kinetics

Abstract

The sulfotransferase (SULT) 2B1b, which catalyzes the sulfonation of 3β-hydroxysteroids, has been identified as a potential target for prostate cancer treatment. However, a major limitation for SULT2B1b-targeted drug discovery is the lack of robust assays compatible with high-throughput screening and inconsistency in reported kinetic data. For this reason, we developed a novel label-free assay based on high-throughput (> 1 Hz) desorption electrospray ionization mass spectrometry (DESI-MS) for the direct quantitation of the sulfoconjugated product (CV < 10 %; < 1 ng analyte). The performance of this DESI-based assay was compared against a new fluorometric coupled-enzyme method that we also developed. Both methodologies provided consistent kinetic data for the reaction of SULT2B1b with its major substrates, indicating the affinity trend pregnenolone > DHEA > cholesterol, for both the phosphomimetic and wild-type SULT2B1b forms. The novel DESI-MS assay developed here is likely generalizable to other drug discovery efforts and is particularly promising for identification of SULT2B1b inhibitors with potential as prostate cancer therapeutics.

Spontaneous Water Radical Cation Oxidation at Double Bonds in Microdroplets

“…The compatibility of DESI-MS with automated high-throughput screening enables chemical analysis at throughputs better than one sample per second using high-density arrays (50 nL samples, up to 6,144 samples per array) ( Morato et al, 2021 ). Applications include screening of organic reactions ( Wleklinski et al, 2018 ; Jaman et al, 2019 , 2020 ; Loren et al, 2019 ; Biyani et al, 2020 ; Ewan et al, 2020 ; Sobreira et al, 2020 ; Li et al, 2021a ; Morato et al, 2021 ), label-free enzymatic assays ( Morato et al, 2020 ; Kulathunga et al, 2022 ), and the generation of spectral libraries ( Le et al, 2021 ). Here, we take advantage of high-throughput DESI to investigate adduct formation by water radical cations in a large compound set.…”

Re‐Imagining Drug Discovery using Mass Spectrometry

“…The use of DESI‐MS for biological assays brings several advantages compared to conventional bioassay approaches as MS is an inherently label‐free technique with high sensitivity and chemical specificity. This not only maximizes the versatility of the platform, reducing the cost and time required to develop new methods for each different substrate or biological target, but it also eliminates safety concerns related to radioactive materials as well as interferences due to the use of non‐native substrates or coupled secondary reactions that are often required to obtain outputs suitable for spectrophotometric determinations [26,32,61,62] . Additionally, the use of DESI in particular as the ionization method allows the direct analysis of complex bioassay mixtures as it has a high tolerance for high concentrations of non‐volatile buffers, salts and detergents, [31,32,62] all typical components of biological assay matrices and commonly not compatible with direct MS analysis.…”

High‐Throughput Diversification of Complex Bioactive Molecules by Accelerated Synthesis in Microdroplets

“…Leveraging this capability and the versatility of automated DESI‐MS for high‐throughput bioanalysis, reaction screening, and small‐scale synthesis, DESI has the potential for accelerating the drug discovery process by iterating through these high‐speed experiments. It is noteworthy that the HT DESI methodology can be extended to bioassays [9f, g] so completing the drug discovery triad of reaction screening, synthesis, and bioactivity measurement [30]…”

“…By taking advantage of the reaction acceleration phenomenon that occurs in DESI microdroplets, extremely rapid reactions can be achieved under appropriate conditions. The speed, small sample sizes, and access to accelerated reactions represented in DESI‐MS facilitate HTE applications as demonstrated by label‐free enzymatic assays, [9f, g] analysis of tissue microarrays, [9h] screening of various reaction conditions, [9c] and small‐scale synthesis [9d, e] . We have previously examined sulfur fluoride exchange (SuFEx) reactions in R‐SO 2 F compounds; [11] here we generate ArO−SO 2 F species and examine highly selective derivatizations of complex molecules allowed by high‐throughput DESI synthesis.…”