Microdroplet Ultrafast Reactions Speed Antibody Characterization

Abstract

Recently, microdroplet reactions have aroused much interest because the microdroplet provides a unique medium where organic reactions could be accelerated by a factor of 103 or more. However, microdroplet reactions of proteins have been rarely studied. We report the occurrence of multiple-step reactions of a large protein, specifically, the digestion, reduction, and deglycosylation of an intact antibody, which can take place in microseconds with high reaction yields in aqueous microdroplets at room temperature. As a result, fast structural characterization of a monoclonal antibody, essential for assessing its quality as a therapeutic drug, can be enabled. We found that the IgG1 antibody can be digested completely by the IdeS protease in aqueous microdroplets in 250 microseconds, a 7.5 million-fold improvement in speed in comparison to traditional digestion in bulk solution (>30 min). Strikingly, inclusion of the reductant tris­(2-carboxyethyl)­phosphine in the spray solution caused simultaneous antibody digestion and disulfide bond reduction. Digested and reduced antibody fragments were either collected or analyzed online by mass spectrometry. Further addition of PNGase F glycosylase into the spray solution led to antibody deglycosylation, thereby producing reduced and deglycosylated fragments of analytical importance. In addition, glycated fragments of IgG1 derived from glucose modification were identified rapidly with this ultrafast digestion/reduction technique. We suggest that microdroplets can serve as powerful microreactors for both exploring large-molecule reactions and speeding their structural analyses.

Enzyme-Catalyzed Hydrolysis of Lipids in Immiscible Microdroplets Studied by Contained-Electrospray Ionization

“…Previous studies have shown increased droplet reactivity with increased flight distance due to solvent evaporation, which leads to concentration and surface effects. − Similar observations have recently been made with the contained-ES apparatus even at short analytical spray distances (<5 mm), , making it possible to accurately examine the kinetics of lipase hydrolysis using known droplet velocities to convert the cavity length to reaction time. This required important control experiments to enable correct interpretation of data.…”

Spontaneous Water Radical Cation Oxidation at Double Bonds in Microdroplets

“…; Wei et al, 2020 , 2022 ; Cao et al, 2020 ; Kuai et al, 2021 ; Vannoy et al, 2021 ; Zheng et al, 2021 ), and the unique properties of the reaction environment ( Li et al, 2014 ; Lee et al, 2019a , 2019b ; Gao et al, 2019 ; Huang et al, 2021 ; Wang et al, 2021 ; Gong et al, 2022 ; Qiu et al, 2022 ). These features give rise to numerous applications ( Wei et al, 2017 ; Yan et al, 2018 ; Zhu et al, 2019 ; Nie et al, 2020 ; Li Z. et al, 2021 ; Gunawardena et al, 2021 ; Yan, 2021 ; Zhang, 2021 ). Reaction acceleration is firmly attributed to the reactions at the solution/air interface ( Yan et al, 2016 ; Lee et al, 2019b ; Wei et al, 2020 ).…”

Microdroplet mass spectrometry: Accelerating reaction and application

“…As depicted in Figure 1C, the droplet reaction with or without charge may be conducted offline and the products were collected and injected onto MS [8,42]. This is particularly useful for sample preparation or large-scale synthesis.…”

“…Microdroplet chemistry has been widely studied using MS mainly via ESI and desorption electrospray ionization (DESI) which are soft ionization methods of MS to transform the analyte molecules in condensed phase to gas phase before entering the MS inlet. Pioneered studies by Cooks [5][6][7] and Zare [8][9][10], noted that when reagents are confined in a microdroplet, various chemical reactions, such as redox reactions [8,[11][12][13][14][15][16], enzyme digestion [8,9], acid-/base-catalyzed reactions [17,18], can occur in microseconds to milliseconds, which is considered 10 5 × faster than the reaction in the bulk. Beyond mechanistic studies, amazing reactions in microdroplets was used in organic synthesis [11,19], chemical derivatization [20], protein folding/unfolding [21], and protein digestion [8,9], which can greatly enhance the capability of application fields including green and sustainable synthesis [11], high-throughput screening [6,12], protein sequencing [8,9], and protein characterization [9].…”