Home List of Titles Rapid fabrication of glass/PDMS hybrid μIMER for high throughput membrane proteomics
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- Rapid fabrication of glass/PDMS hybrid μIMER for high throughput membrane proteomics
- Pereira-Medrano, Ana G.; Forster, Simon; Fowler, Gregory J. S.; McArthur, Sally L.; Wright, Phillip C.
- Mass spectrometry (MS) based proteomics has brought a radical approach to systems biology, offering a platform to study complex biological functions. However, key proteomic technical challenges remain, mainly the inability to characterise the complete proteome of a cell due to the thousands of diverse, complex proteins expressed at an extremely wide concentration range. Currently, high throughput and efficient techniques to unambiguously identify and quantify proteins on a proteome-wide scale are in demand. Miniaturised analytical systems placed upstream of MS help us to attain these goals. One time-consuming step in traditional techniques is the in-solution digestion of proteins (4–20 h). This also has other drawbacks, including enzyme autoproteolysis, low efficiency, and manual operation. Furthermore, the identification of ∝-helical membrane proteins has remained a challenge due to their high hydrophobicity and lack of trypsin cleavage targets in transmembrane helices. We demonstrate a new rapidly produced glass/PDMS micro Immobilised Enzyme Reactor (μIMER) with enzymes covalently immobilised onto polyacrylic acid plasma-modified surfaces for the purpose of rapidly (as low as 30 s) generating peptides suitable for MS analysis. This μIMER also allows, for the first time, rapid digestion of insoluble proteins. Membrane protein identification through this method was achieved after just 4 min digestion time, up to 9-fold faster than either dual-stage in-solution digestion approaches or other commonly used bacterial membrane proteomic workflows.
- Publication type
- Journal article
- Research centre
- Swinburne University of Technology. Faculty of Engineering and Industrial Sciences
- Lab on a Chip, Vol. 10, no. 24 (2010), pp. 3397-3406
- Publication year
- High throughput; Proteomics; Systems biology
- Royal Society of Chemistry
- Publisher URL
- Copyright © The Royal Society of Chemistry 2010. The authors give the Royal Society of Chemistry the exclusive right and licence throughout the world to edit, adapt, translate, reproduce and publish the Paper in all formats, in all media and by all means (whether now existing or in future devised). Published version of the paper reproduced here for non-commercial purposes only in accordance with the copyright policy of the publisher. This paper is made available for personal use only; no further reuse is permitted.
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