Sample Stacking Provides Three Orders of Magnitude Sensitivity Enhancement in SDS Capillary Gel Electrophoresis of Adeno-Associated Virus Capsid Proteins

Abstract

Size-based protein analysis utilizing only 25 ng of total proteins has been realized by sodium dodecyl sulfate capillary gel electrophoresis (SDS CGE) with head-column field-amplified sample stacking as an online sample preconcentration technique. This method has been used as a replacement of SDS-PAGE for purity analysis of adeno-associated virus (AAV) therapeutic products of different serotypes and transgenes. A limit of detection of 0.2 ng/mL (3.3 pM) capsid proteins was achieved with convenient UV absorbance detection at 214 nm, equivalent to 20 pg of protein (330 attomole) loaded in the autosampler vial. For purity analysis, only 25 ng of total AAV capsid proteins (4.3 femtomole virus particles) were loaded to the autosampler vial. The sensitivity is comparable to silver-stained SDS-PAGE. The RSD of purity measurement was 0.0-0.8%, comparable to conventional SDS CGE utilizing 0.1-0.5 mg proteins. The new method provided 3 orders of magnitude sensitivity enhancement as compared to conventional SDS CGE. It shares all the advantages of conventional SDS CGE (labor-saving, easy automation, and convenient quantitation) and also the high sensitivity of silver stained SDS-PAGE. The sample stacking SDS CGE technique can be adopted for size-based analysis of other types of proteins. It is especially useful when protein quantity or concentration is not sufficient for regular SDS CGE or SDS-PAGE assay.

Recent advances in enhancing the sensitivity of electrophoresis and electrochromatography in capillaries and microchips (2016–2018)

“…Zhang and Meagher reported three orders of magnitude sensitivity improvement by using FASI in SDS‐CGE of adeno‐associated virus (AAV) capsid proteins. The LOD of 0.2 ng/mL was achieved with conventional UV detection.…”

“…FASS and FASI are usually the first choice among all stacking techniques due to their simplicity and high compatibility with different CE modes such as CZE , EKC , CGE , CEC as well as microchip electrophoresis . An uncomplicated buffer system used in FASS and FASI allows to utilize a whole range of detectors including UV , LED‐IF , LIF , C4D , and amperometric .…”

“…FASS and FASI are usually the first choice among all stacking techniques due to their simplicity and high compatibility with different CE modes such as CZE , EKC , CGE , CEC as well as microchip electrophoresis . An uncomplicated buffer system used in FASS and FASI allows to utilize a whole range of detectors including UV , LED‐IF , LIF , C4D , and amperometric . The sample matrix effect is reduced or eliminated since the sample clean‐up is necessary to lower the conductivity of the sample and thus, FASS and FASI can be used in analysis of biological , pharmaceutical , environmental , food , industrial , and forensic samples .…”

“…An uncomplicated buffer system used in FASS and FASI allows to utilize a whole range of detectors including UV , LED‐IF , LIF , C4D , and amperometric . The sample matrix effect is reduced or eliminated since the sample clean‐up is necessary to lower the conductivity of the sample and thus, FASS and FASI can be used in analysis of biological , pharmaceutical , environmental , food , industrial , and forensic samples . In addition, combination of FASS and FASI with other stacking techniques is popularly used in multi‐stacking approaches which will be discussed further in this review.…”

Recent advances in microscale separation

“…Zhang et al. coupled CGE with an online sample preconcentration technique. Size‐based protein analysis utilizing only 25 ng of total proteins was realized.…”