Rapid fabrication of glass/PDMS hybrid µIMER for high throughput membrane proteomics.
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 α-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.
Biological and Environmental Systems Group, ChELSI Institute, Chemical and Biological Engineering, The University of Sheffield, Mappin Street, Sheffield, S1 3JD, UK.
Ana G
AG
Pereira-Medrano
Simon
S
Forster
Gregory J S
GJ
Fowler
Sally L
SL
McArthur
Phillip C
PC
Wright
D000180:Acrylic Resins / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry; D004129:Dimethylpolysiloxanes / Q000737:chemistry*; D004798:Enzymes / Q000737:chemistry; D005898:Glass / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods; D008565:Membrane Proteins / Q000737:chemistry*; D046228:Microarray Analysis / Q000295:instrumentation* / Q000379:methods; D010455:Peptides / Q000737:chemistry; D017433:Protein Structure, Secondary; D020543:Proteome; D040901:Proteomics / Q000379:methods*; D046939:Synechocystis / Q000378:metabolism; D013997:Time Factors; D014357:Trypsin / Q000737:chemistry
D000180:Acrylic Resins; D001426:Bacterial Proteins; D004129:Dimethylpolysiloxanes; D004798:Enzymes; D008565:Membrane Proteins; D010455:Peptides; D020543:Proteome; C006903:carbopol 940; C013830:baysilon; D014357:Trypsin