Size‐based protein separations by microchip electrophoresis using an acid‐labile surfactant as a replacement for SDS

Abstract

We demonstrate the use of an acid-labile surfactant (ALS) as a replacement for SDS for size-based protein separations in a microfluidic device. ALS is of interest to the proteomic field as it degrades at low pH and hence can be removed to reduce surfactant interference with down-stream MS. A range of SDS and ALS concentrations were tested as denaturants for microchip electrophoresis to investigate their effects on the separation of proteins from 18 to 116 kDa and to provide a suitable comparison between the two surfactants. The electrophoretic mobilities of the proteins were not significantly affected by the use of ALS instead of SDS. Protein separations with ALS are performed in less than 3 min, which is a significant decrease in the time compared with the previous ALS separations on a slab gel format. We also demonstrate the use of poly-N-hydroxyethylacrylamide as a dynamic, hydrophilic chip channel coating that can be applied with a rapid and simple protocol for size-based protein separation. The results reported here could significantly decrease the time and increase the attainable level of automation and integration of the front-end protein fractionation required for "top-down" proteomics.

Polymer sieving matrices in microanalytical electrophoresis

“…Dynamic coatings can be replenished by replacing the background buffer, so that run-to-run reproducibility is excellent. 54 Reagents for this purpose include polymers of acrylamide 60,80 and acrylamide derivatives such as PDMA 34 and PHEA (poly-N-hydroxyethylacrylamide), 81 polysaccharide and its derivatives such as dextran, 82 HPMC, 56 MC, 83 HEC, 53,84,85 HPC, 86 cationic HEC, 87 MHEC (methylhydroxyethylcellulose), 36 cationic starch derivatives 88 and other polymers such as PVP, 89 PVA (polyvinyl alcohol) 90 and PEG (polyethylene glycol). 91 For detail, readers are directed to the recent reviews by Horvath and Dolník, 83 Dolník, 91 Doherty et al, 81 and Belder and Ludwig.…”

“…Dynamic coatings can be replenished by replacing the background buffer, so that run-to-run reproducibility is excellent. 54 Reagents for this purpose include polymers of acrylamide 60,80 and acrylamide derivatives such as PDMA 34 and PHEA (poly-N-hydroxyethylacrylamide), 81 polysaccharide and its derivatives such as dextran, 82 Lastly, novel sieving matrices that offer complex properties are being more widely adopted. These include: thermally responsive polymers, 93 interpenetrating networks (IPN) 94 and carbon nanotube (CNT) modied polymers (see chapter 5 also for more detail).…”

Recent innovations in protein separation on microchips by electrophoretic methods: An update

“…Root et al . recently performed size‐based protein separation with an acid‐labile surfactant (ALS) on a glass microchip dynamically coated with poly‐( N ‐hydroxyethylacrylamide) polymer 75. Proteins from 18 to 116 kDa in size were separated with high resolution with ALS and SDS for comparison.…”

“…Root et al 75 recently reported the use of an ALS as a replacement for SDS for size‐based protein separations on microfluidic chips. ALS is of particular interest in proteomics, as it breaks down at low pH and can therefore be removed to reduce surfactant interference with downstream MS analysis.…”

Simplified Microchip Electrophoresis for Rapid Separation of Urine Proteins

“…ME has been demonstrated to be a powerful tool for efficient separation of DNA fragments (13,14), amino acids (15,16), and drugs (17). As far as protein analysis is concerned, ME separation of proteins is currently limited to purified proteins (18)(19)(20)(21). There is no report concerning using microfluidic chip to separate urine proteins.…”