Digital microfluidic hydrogel microreactors for proteomics

Proteomics
Vivienne N LukAaron R Wheeler

Abstract

Proteolytic digestion is an essential step in proteomic sample processing. While this step has traditionally been implemented in homogeneous (solution) format, there is a growing trend to use heterogeneous systems in which the enzyme is immobilized on hydrogels or other solid supports. Here, we introduce the use of immobilized enzymes in hydrogels for proteomic sample processing in digital microfluidic (DMF) systems. In this technique, preformed cylindrical agarose discs bearing immobilized trypsin or pepsin were integrated into DMF devices. A fluorogenic assay was used to optimize the covalent modification procedure for enzymatic digestion efficiency, with maximum efficiency observed at 31 μg trypsin in 2-mm diameter agarose gel discs. Gel discs prepared in this manner were used in an integrated method in which proteomic samples were sequentially reduced, alkylated, and digested, with all sample and reagent handling controlled by DMF droplet operation. Mass spectrometry analysis of the products revealed that digestion using the trypsin gel discs resulted in higher sequence coverage in model analytes relative to conventional homogenous processing. Proof-of-principle was demonstrated for a parallel digestion system in which a si...Continue Reading

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Citations

Apr 10, 2013·Analytical and Bioanalytical Chemistry·Yong Zeng, Tanyu Wang
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Nov 13, 2012·Analytical Chemistry·Michelle L KovarikNancy L Allbritton
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Jun 5, 2012·Biomicrofluidics·Lindsey K FiddesAaron R Wheeler
Feb 26, 2014·Nature Communications·Irwin A EydelnantAaron R Wheeler
Apr 25, 2012·Annual Review of Analytical Chemistry·Kihwan ChoiAaron R Wheeler
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May 1, 2021·Nanomaterials·Rui VitorinoVáclav Kašička
Apr 9, 2019·Langmuir : the ACS Journal of Surfaces and Colloids·Ian SwyerAaron R Wheeler

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