In-Source Microdroplet Derivatization Using Coaxial Contained-Electrospray Mass Spectrometry for Enhanced Sensitivity in Saccharide Analysis

Abstract

Online, droplet-based in-source chemical derivatization is accomplished using a coaxial-flow contained-electrospray ionization (contained-ESI) source to enhance sensitivity for the mass spectrometric analysis of saccharides. Derivatization is completed in microseconds by exploiting the reaction rate acceleration afforded by electrospray microdroplets. Significant improvements in method sensitivity are realized with minimal sample preparation and few resources when compared to traditional benchtop derivatizations. For this work, the formation of easily ionizable phenylboronate ester derivatives of several mono-, di-, and oligosaccharides is achieved. Various reaction parameters including concentration and pH were evaluated, and a Design of Experiments approach was used to optimize ion source parameters. Signal enhancements of greater than two orders of magnitude were observed for many mono-and disaccharides using in-source phenylboronic acid derivatization, resulting in partsper-trillion (picomolar) limits of detection. In addition, amino sugars such as glucosamine, which do not ionize in negative mode, were detected at low parts-per-billion concentrations, and isobaric sugars such as lactose and sucrose were easily distinguished. The new in-source derivatization approach can be employed to expand the utility of ESI-MS analysis for compounds that historically experience limited sensitivity and detectability, while avoiding resource-intensive, bulk-phase derivatization procedures.

Liquid Chromatography–Tandem Mass Spectrometry with Online, In-Source Droplet-Based Phenylboronic Acid Derivatization for Sensitive Analysis of Saccharides

“…With the exception of sucrose and raffinose, the underivatized sugars did not generate abundant product ions and so SIM acquisition was the more sensitive mode of analysis. Any differences in sensitivity attributed to the use of the two different ion sources (contained-ESI vs conventional ESI) are likely minimal, as our previous work using only the contained-ESI source established that the sensitivity gains are due to the derivatization reaction rather than the type of source used …”

“…MS analysis was conducted using a Thermo Finnigan LTQ linear ion trap mass spectrometer (Thermo Scientific, San Jose, CA) operating in the negative-ion mode. Mass spectrometer inlet temperature, sheath gas pressure, and source voltage were optimized previously . The final source conditions were as follows: sheath gas pressure ( N 2 ) = 0–5 psi, source voltage = −6.5 kV, capillary temperature = 325 °C, reagent concentration = 4 mM, reagent flow rate = 5 μL/min, and LC eluent flow rate = 75 μL/min.…”

“…LODs for the native, underivatized analytes were determined in a similar fashion except using the commercial HESI ion source. Our previous work revealed that monitoring the deprotonated molecular ion [M – H] − in the selected ion monitoring (SIM) mode was more sensitive for most of the underivatized sugars than using MS/MS . Thus, for all underivatized sugars except sucrose and raffinose, the SIM mode was used to determine LOD/LOQ.…”

“…In previous works, we leveraged the rate acceleration afforded by electrospray microdroplets to carry out real-time chemical derivatization of saccharides directly within the ion source of a mass spectrometer during the ionization process . This was accomplished using a unique, coaxial flow contained-electrospray ion source (contained-ESI) previously developed in our lab .…”

Investigation of Tryptic Protein Digestion in Microdroplets and in Bulk Solution

“…Numerous studies in the past decade have shown that reaction rates can be dramatically accelerated if reactions are conducted in microdroplet rather than in bulk solution. − The reaction acceleration has mainly been attributed to the special reaction environment at the air–liquid surface of microdroplet where partial solvation of the reagents, the presence of a strong electric field, enhanced concentration of solute, rapid desolvation, and surface excess charge accumulation could be the influencing factors to speed up the reaction. − This “catalytic” feature allows microdroplet chemistry to have impactful applications including rapid chemical derivatization, − reaction kinetic studies, high-throughput reaction screening, nanomaterial preparation, − and the rapid, green and sustainable synthesis or degradation. ,− However, most of these reactions involved small organic molecules, large molecule protein reactions in microdroplets are rare, except enzymatic digestion of proteins. − The microdroplet digestion of proteins is very fast and completes in submillisecond time scale (e.g., within 250 μs). − …”