Development and characterization of immobilized enzymatic reactors for on-line comprehensive bottom-up proteomics

Research output: Unpublished contribution to conferencePoster

Abstract

Conventional comprehensive proteomics workflows suffer from significant variation in sample preparation due to excessive pipetting and vessel changes during biochemical modifications. A number of techniques have been
developed to combat this variation, including, one-pot sample preparation[1], novel integrated fluidic setups (iPAD)[2] and oil-air droplet systems (OAD)[3]. A major disadvantage of these techniques is the multiple handling
steps necessary to add different reagents which ultimately will impact sample loss, additionally, the slow kinetic performance of proteolysis leads to multi-hour protein digestion times.
Immobilized enzymatic reactors (IMERs) provide a facile methodology to address both the variation as well as the slow digestion times in global proteomics studies. IMER technology involves the covalent attachment of bioactive
proteins to a framework which allows for ultrafast protein digestion (minute to second scale) by catalyzing the proteolysis of proteins.
Monolithic frameworks represent a particularly apt format to produce IMERs due to the tunable porosity which can allow for optimal surface area to porosity ratio to efficiently immobilize bioactive enzymes in comparison to
open tubular columns, as well as low backpressure allowing faster flowrates and subsequent digestion speeds in comparison to packed particle columns.
Herein, we present the development of novel on-line IMER LC-MS workflow optimized for minimal peptide carryover and digestion reproducibility. A tertiary monomeric polymer was produced in a single porogenic solvent to produce
monoliths inside 100 μm I.D capillaries capable of being integrated into nanoLC-MS setups. We optimized the hydrophobicity to reduce non-specific hydrophobic interactions to decrease sample carryover as well as fine-tuned the maximal concentration immobilized trypsin enzyme.
In this way it was possible to comprehensively characterize complex proteome samples in less than 1 hour with good repeatability. The beforementioned IMERs represent a suitable technology to be seamlessly integrated into current
nanoLC-MS setups, removing the need for conventional sample preparation methods.

[1] Y. Cong et al., “Improved Single-Cell Proteome Coverage Using Narrow-Bore Packed NanoLC Columns and Ultrasensitive Mass Spectrometry,” Anal. Chem., 92, 2020, 2665–2671.
[2] X. Shao et al., “Integrated Proteome Analysis Device for Fast Single-Cell Protein Profiling,” Anal. Chem., 90, 2018, 14003–14010.
[3] Z.Y. Li et al., “Nanoliter-Scale Oil-Air-Droplet Chip-Based Single Cell Proteomic Analysis,” Anal. Chem., 90, 2018, 5430–5438.
Original languageEnglish
Publication statusPublished - May 2022
Event17th International Symposium on Hyphenated Techniques in Chromatography and Separation Technology - Ghent, Belgium
Duration: 18 May 202220 May 2022
Conference number: 17
https://htc-17.com/

Conference

Conference17th International Symposium on Hyphenated Techniques in Chromatography and Separation Technology
Abbreviated titleHTC-17
Country/TerritoryBelgium
CityGhent
Period18/05/2220/05/22
Internet address

Keywords

  • IMER
  • Comprehensive global proteomics
  • Sample preparation
  • LC-MS

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