Materials and Methods
ATTO-TAG FQ and potassium cyanide which are included in ATTO-TAG™ FQ Amine-Derivatization Kit (PN A2334) were purchased from ThermoFisher Scientific (Waltham, MA, U.S.A.). Methanol (PN 1.06018.2500) was obtained from VWR (Radnor, PA, U.S.A.). Sodium dodecyl sulfate (SDS) (PN L4390-100G) and N-Ethylmaleimide (NEM) (PN E3876-5G) were obtained from Sigma-Aldrich (St. Louis, MO, U.S.A.). Amicon Ultra-0.5 Centrifugal Filters with 10,000 NMWL (PN UFC501024) were purchased from EMD Millipore (Billerica, MA, U.S.A.). The SDSMW Analysis Kit (PN 390953) (Figure 1B) was from SCIEX (Framingham, MA, U.S.A.), which includes the SDS-MW gel buffer(proprietary formulation, pH 8, 0.2% SDS), acidic wash solution (0.1 N HCl), basic wash solution (0.1 N NaOH) and the SDS-MW sample buffer(100 mM Tris-HCl pH 9.0, 1%SDS). EZCE Capillary Cartridge (PN A55625, SCIEX, Framingham, MA, U.S.A) pre-assembled with bare fused-silica capillary (50 µm I.D., 30 cm total length, 20 cm effective length) was used for separation (Figure 1C). Universal vials (PN A62251), universal vial caps (PN A62250) and PCR vials (PN 144709) from SCIEX (Framingham, MA, U.S.A.) were used for sample solution loading.
A PA 800 Plus Pharmaceutical Analysis CE system (SCIEX, Framingham, MA, U.S.A.) equipped with a laser-induced fluorescence (LIF) detector with a 600nm emission filter (Figure 1A) was used for all the experiments.
Data acquisition and analysis were performed using 32 KaratTM Software 10.
Packaged AAV2 of pAV-CMV-GFP with titer at 2.24X1013GC/mL (titer as supplied by vendor), packaged AAV8 of pAV-CMV-GFP with titer at 1.57 X1014GC/mL (titer as supplied by vendor) and formulation buffer (Phosphate Buffered Saline, pH 7.5 with 0.001% pluronic F68) were purchased from Vigene Biosciences (Rockville, MD, U.S.A.). Both samples were kept in formulation buffer.
4%SDS in 150 mM NEM solution. Weigh 93.75mg NEM and 0.2g SDS in 5mL DI water. Mix to dissolve.
Labeling Solution. The 20 mM FQ dye stock solution was prepared in methanol. This solution was further diluted in DDI water to a final concentration of 2.5 mM and was used as the FQ dye working solution. The nucleophile reagent used was potassium cyanide at a concentration of 30 mM.
1%SDS solution. Dissolve 1g of SDS in 100mL DI water.
Sample Preparation Procedure. 10µL of AAV8 sample solution was mixed with 1.2µL of 4% SDS in 150mM NEM solution in a 0.65 mL micro-centrifuge tube and incubated at 70°C for 5 minutes. Then, the sample solution was mixed with 1.5 µL of 2.5mM FQ dye working solution and 1 µL of 30mM KCN solution and incubated at 70°C for 10minutes. 28 µL of 1% SDS was added to quench the labeling reaction. The sample solution was incubated at 70°C for 5 minutes. At last, 20µL of DI water was added to the mixture. The diluted mixture was transferred to the sample vial for analysis on the PA800 Plus. 5
Buffer exchange or desalting to lower final salt concentration is an optional step before sample solution is loaded into sample vials to improve sensitivity of the method due to less competition from the salt during electrokinetic injection. The mixture of 5% of 1%SDS, 1.25% of formulation buffer in DI water was used as buffer exchange solution in this technical note.
The user guide of the SDS-MW Analysis kit was followed for instrument setup. Water plug was used in separation method for online sample concentration to improve the sensitivity of the method. 2,3,4
The “Initial Conditions” and “LIF Detector Initial Conditions” were set up as indicated in Figure 3 and Figure 4, respectively. Same initial setup was used for conditioning, separation and shutdown methods.