Fast protein analysis enabled by high-temperature hydrolysis

Wang, Yuchen; Zhang, Wenpeng; Ouyang, Zheng

doi:10.1039/d0sc03237a

Cited by 13 publications

(11 citation statements)

References 51 publications

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“…However, digestion is usually a time-consuming step that can take from 30 min to overnight incubation. , In addition, commonly used digestion methods often include additional steps of protein denaturation, reduction, and alkylation to unfold the mAb structure and facilitate digestion; these additional steps may also lengthen the process time and reduce the analysis speed and throughput . To accelerate digestion of mAb (or other proteins), a variety of methods have been investigated, including increasing the digestion temperature, adding organic solvents, applying microwave energy, using high-intensity focused ultrasound, or employing a microchip reactor. − Nevertheless, an alternative method that is very fast for mAb digestion would be highly valuable.…”

Section: Introductionmentioning

confidence: 99%

Microdroplet Ultrafast Reactions Speed Antibody Characterization

et al. 2021

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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.

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Section: Introductionmentioning

confidence: 99%

Microdroplet Ultrafast Reactions Speed Antibody Characterization

et al. 2021

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“…As another example, POC analysis of protein biomarkers, such as ESAT-6, was performed by coupling a high-temperature micro-reactor with a miniature dual-LIT MS system ( Fig. 5 f) [ 90 ]. The ESAT-6 is a biomarkers of Mycobacterium tuberculosis (Mtb) infections for the diagnosis of tuberculosis (TB) disease.…”

Section: Ms Miniaturization: a Necessary Step To On-site Applicationsmentioning

confidence: 99%

Recent advances in on-site mass spectrometry analysis for clinical applications

Zhou¹,

Zhang²,

Ouyang³

2022

TrAC Trends in Analytical Chemistry

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In recent years, mass spectrometry (MS) is increasingly attracting interests for clinical applications, which also calls for technical innovations to make a transfer of MS from conventional analytical laboratories to clinics. The system design and analysis procedure should be friendly for novice users and appliable for on-site clinical diagnosis. In addition, the analysis result should be auto-interpreted and reported in formats much simpler than mass spectra. This motivates new ideas for developments in all the aspects of MS. In this review, we report recent advances of direct sampling ionization and miniature MS system, which have been developed targeting clinical and even point-of-care analysis. We also discuss the trend of the development and provide perspective on the technical challenges raised by diseases such as coronavirus SARS-CoV-2.

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“…To fully exploit this advantage for high-throughput proteomic workflows, attempts have been undertaken to accelerate the method using microwave radiation [19][20][21][22] and increasing the temperature. [23][24][25] Yet, no method based on acid cleavage of proteins has gained grounds in proteomics, arguably because the time savings were still insufficient and/or the workflows remained as laborintensive as in standard trypsin digestion. Moreover, no method truly allowed its simple online integration to LC-MS with the possibility of separating the cleavage products because sophisticated accessories not broadly available to other researchers were required.…”

Section: Introductionmentioning

confidence: 99%

Easy, robust, and repeatable online acid cleavage of proteins in mobile phase for fast quantitative LC-MS bottom-up proteomics – application for ricin detection

Naplekov

Jadeja

Fučíková

et al. 2023

Preprint

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Sample preparation involving the cleavage of proteins in peptides is the first critical step for successful bottom-up proteomics analyses. The main limitations to commonly used methods for protein sample preparation are time- and labor-intensiveness. Here we report a fast online method for post-injection acid cleavage of proteins directly in the mobile phase typically used for LC-MS analyses in proteomics. The chemical cleavage is achieved in 0.1% formic acid within 35 s in a capillary heated to 195 °C installed upstream of the analytical column, enabling the generated peptides to be separated. The peptides generated by the optimized method covered the entire sequence except for one amino acid of trastuzumab used for the method development. The qualitative results are extraordinarily stable, even over a long period of time. Moreover, the method is also suitable for accurate and repeatable quantification. The procedure requires only one manual step, significantly decreasing sample transfer losses. To demonstrate its practical utility, we tested the method for the fast detection of ricin. Ricin can be unambiguously identified from an injection of 10 ng, and the results can be obtained within 7-8 min after receiving a suspicious sample. Because no sophisticated accessories and no additional reagents are needed, the chemical method can be seamlessly transferred to any laboratory for high-throughput proteomic workflows.

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Fast protein analysis enabled by high-temperature hydrolysis

Abstract: While the bottom-up protein analysis serves as a mainstream method for biological studies, its efficiency is limited by the time-consuming process for enzymatic digestion or hydrolysis as well as the...

Cited by 13 publications

References 51 publications

Microdroplet Ultrafast Reactions Speed Antibody Characterization

Microdroplet Ultrafast Reactions Speed Antibody Characterization

Recent advances in on-site mass spectrometry analysis for clinical applications

Easy, robust, and repeatable online acid cleavage of proteins in mobile phase for fast quantitative LC-MS bottom-up proteomics – application for ricin detection

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