A CIEF‐LIF method for simultaneous analysis of multiple protein kinases and screening of inhibitors

Abstract

Here, a CIEF‐LIF method for multiple protein kinase simultaneous analysis and inhibitors throughput screening with fast rate and low cost is presented. Comparing with CZE, CIEF‐LIF exhibited great focusing ability and high separation efficiency for substrate and phosphorylated peptides, and is applicable for multiple kinases simultaneous analysis regardless of their substrate peptides compositions and charge statuses. Thus, highly sensitive analysis for cyclic adenosine 3’, 5’‐monophosphate‐dependent protein kinase (PKA) and cyclin‐dependent kinase 1 (CDK1) was achieved in CIEF‐LIF analysis with detection sensitivity up to 1.25 mU/μL and 0.4 mU/μL, respectively, two magnitudes higher than that of CZE and comparable with that in nanomaterials or green fluorescent protein‐based kinase assay. Moreover, the inhibition effect of inhibitors on multiple kinases could be simultaneously readout in a single electrophoretic run, with half maximal inhibitory concentration of H‐89 for PKA and Ro‐3306 for CDK1 calculated as 37.0 and 35.9 nM, respectively, consistent with literatures reported. The CIEF‐LIF also exhibited strong anti‐interference ability in human breast cancer cell lysates analysis and simulators such as forskolin and 3‐isobutyl‐1‐methylxantine assessment. Therefore, CIEF‐LIF is desirable for future biological application and clinical diagnostics and drug discovery.

Capillary Electrophoresis: Trends and Recent Advances

“…In a similar work, cIEF-LIF was compared to CZE for its ability to simultaneously study composition and inhibition of multiple protein kinases. 89 The method was successful regardless of structure and charge of the substrate peptides. The use of nanogels to physically constrain an enzyme in a separation capillary while performing electrophoretically mediated microanalysis (EMMA) was proposed for improving sensitivity and separation specificity.…”

Advances in screening enzyme inhibitors by capillary electrophoresis

“…Online enzyme assay integrates together enzyme incubation, reaction, and product detection in a single capillary within one run. Comparing off‐line enzyme assay, it thus greatly simplified enzyme assay process, and has developed two mainly operation modes, at inlet and electrophoretically mediated microanalysis (EMMA) .…”

Recent advances in CE and microchip‐CE in clinical applications: 2014 to mid‐2017

“…The use of CE and ME in clinical protein analysis has not shown the expected growth that was expected. There have been some advances in the types of detectors used such as CL , LIF , LED‐LIF , and MS . An interesting study on protein analysis in the vitreous humor of the eye was reported by Reich et al.…”