Quantifying Carbohydrate-Active Enzyme Activity with Glycoprotein Substrates Using Electrospray Ionization Mass Spectrometry and Center-of-Mass Monitoring

Abstract

Carbohydrate-active enzymes (CAZymes) play critical roles in diverse physiological and pathophysiological processes and are important for a wide range of biotechnology applications. Kinetic measurements offer insight into the activity and substrate specificity of CAZymes, information that is of fundamental interest and supports diverse applications. However, robust and versatile kinetic assays for monitoring the kinetics of intact glycoprotein and glycolipid substrates are lacking. Here, we introduce a simple but quantitative electrospray ionization mass spectrometry (ESI-MS) method for measuring the kinetics of CAZyme reactions involving glycoprotein substrates. The assay, referred to as center-of-mass (CoM) monitoring (CoMMon), relies on continuous (real-time) monitoring of the CoM of an ensemble of glycoprotein substrates and their corresponding CAZyme products. Notably, there is no requirement for calibration curves, internal standards, labeling, or mass spectrum deconvolution. To demonstrate the reliability of CoMMon, we applied the method to the neuraminidase-catalyzed cleavage of N-acetylneuraminic acid (Neu5Ac) residues from a series of glycoproteins of varying molecular weights and degrees of glycosylation. Reaction progress curves and initial rates determined with CoMMon are in good agreement (initial rates within ≤5%) with results obtained, simultaneously, using an isotopically labeled Neu5Ac internal standard, which enabled the time-dependent concentration of released Neu5Ac to be precisely measured. To illustrate the applicability of CoMMon to glycosyltransferase reactions, the assay was used to measure the kinetics of sialylation of a series of asialo-glycoproteins by a human sialyltransferase. Finally, we show how combining CoMMon and the competitive universal proxy receptor assay enables the relative reactivity of glycoprotein substrates to be quantitatively established.

Native Mass Spectrometry Quantitation of α2-3-Linked N-Acetylneuraminic Acid Content of Prostate-Specific Antigen: An Accurate Liquid Biopsy for Clinically Significant Prostate Cancer

“…perfringens (a non-linkage specific neuraminidase) prior to ESI-MS analysis . The weighted-average MW or center-of-mass (CoM) of PSA without enzymatic treatment (CoM 0 ), with treatment with NEUS (CoM S ), and with treatment with NEUC (CoM ∞ ) were determined from the corresponding mass spectra using CoM monitoring (CoMMon), a technique described elsewhere . The %α23PSA is then calculated from the ratio of the change in CoM upon treatment with NEUS and treatment with NEUC (eq S2d).…”

“…Details of the CUPRA-ZYME and CoMMon methods are given as Supporting Information and described in detail elsewhere. , Briefly, the CUPRA-ZYME method relies on continuous monitoring of the concentration of a CUPRA substrate (CS) and corresponding product (CP) using native MS. The CS and CP concentrations were calculated (eqs S1a–c) from the relative abundances of CS and CP bound to a universal proxy protein ( Uni P proxy ≡ hCA), as measured by ESI-MS. , Progress curves (fractional abundance of product CP vs time, f CP,t ) were constructed from the time-resolved ESI mass spectra (eq S1c). The time-dependent fraction ( f t ) of Neu5Ac associated with PSA N -glycans during neuraminidase-catalyzed hydrolysis was calculated (eq S2a) from the absolute ratio of the difference in the initial center-of-mass (CoM 0 ) of the PSA at time t (CoM t ) and the difference between CoM 0 and CoM at completion of the reaction (CoM ∞ ).…”

“…Cytidine-5′-monophosphate- N -acetylneuraminic acid (CMP-Neu5Ac) was purchased from Sigma-Aldrich Canada (Oakville, Canada) and N -acetyl-neuraminic acid-1,2,3- 13 C 3 (Neu5Ac- 13 C 3 ) was purchased from Omicron Biochemicals Inc. (South Bend, IN, USA). Neu5Acα2-3Galβ1-4GlcNAc-ethylamine (3SLNAc-ethylamine) and the CUPRA substrates CS 3SL , CS 6SL , and CS 3SLNAc , produced from the oligosaccharides 3SL, 6SL, and 3SLNAc, respectively, and containing a sulfonamide affinity tag (Figure S1), were synthesized, as described elsewhere. , …”

“…Center-of-mass (CoM) values were calculated automatically from the average mass spectra using the SWARM software (https://github.com/pkitov/CUPRA-SWARM) and used to construct reaction progress curves. 42,43 NEUS Specificity. The specificity of NEUS for the PSA Nglycans with α2-3and α2-6-Neu5Ac was quantified using native MS and CoMMon, combined with the time-dependent Competitive Universal Proxy Receptor Assay (CUPRA-ZYME).…”

“…Cytidine-5′-monophosphate-Nacetylneuraminic acid (CMP-Neu5Ac) was purchased from Sigma-Aldrich Canada (Oakville, Canada) and N-acetylneuraminic acid-1,2,3-13 C 3 (Neu5Ac- 13 respectively, and containing a sulfonamide affinity tag (Figure S1), were synthesized, as described elsewhere. 42,43 PSA Extraction. PSA from blood serum was extracted using biotinylated anti-PSA mAb following previously reported protocols with modifications.…”