Rapid Sizing of Proteins and Antibodies

Transcription

1 Rapid Sizing of Proteins and Antibodies Keywords: Hydrodynamic radius, diffusion coefficient, sizing, Taylor dispersion analysis, protein, antibodies, quality control, protein folding Summary Proteins and Antibodies are vital to our understanding of the human body and understanding them better allows for further advances in all aspects of medicine and medical treatment. Having the ability to test small amounts of proteins and antibodies at low and high concentrations, enables scientists to work with their samples in their native format, without the need to prepare the sample which could lead to changing nature of the sample. Paraytec have developed a new approach to determining the hydrodynamic radius of species in solution. This application note shows the applicability of the ActiPix TDA200 HT Nano-Sizing System for determination of hydrodynamic radii of protein and antibody samples. Excellent correlation was obtained between experimental and expected values with typical analysis times of less than 10 minutes. Figure 1. ActiPix TDA200 Nano-Sizing System Figure 2. ActiPix Nano-Sizing Cartridge Figure 2. Illustration of the change in peak profile as due to Taylor Dispersion as a slug of sample is pushed through a capillary Paraytec Ltd 1

2 Summary of Paraytec s Approach Paraytec have developed a system for rapid determination of protein radii known as the ActiPix TDA200 HT Nano-Sizing System (Figure 1). A detailed application note summarizing this technique 1 can be downloaded from Paraytec s website. For the purposes of this application note, a brief summary of the technique is given below. The ActiPix HT Nano-Sizing System is a high precision nano-sizing system consisting of a precision nano-injector, autosampler and detector. Samples are typically stored in the autosampler prior to injection of a few nanolitres of each sample into a fused capillary of outer diameter typically 360 µm. A plug of the sample, typically nl, is injected at the capillary inlet of a specially designed sizing cartridge (see Figure 3) and driven by application of external pressure along the capillary. UV absorption of the sample is recorded in the first and the second detection window using the ActiPix D100 detector. Whilst the area of the peak is the same, the widths of both peaks are different: the signal from the second window has a greater width and lower amplitude due to Taylor dispersion. The peaks are fitted with an appropriate peak fitting function using software supplied with the system. The area under the peak corresponds to the amount of the sample injected. The standard deviations are used to calculate the hydrodynamic radius of the sample. Theory behind Taylor Dispersion Analysis Band broadening due to Taylor dispersion has been well characterized in previous literature. 1,2 In this method, the absorbance versus time data is processed to obtain the peak centre times at the first and second window, t 1 and t 2 respectively, and the corresponding standard deviations, 1 and 2. These values are used to calculate the hydrodynamic radius, R h, using the equation below R 4k T r t t h B Figure 4. Example of a single Gaussian peak fitted by Paraytec Software 2011 Paraytec Ltd 2

3 Experimental Procedure Samples are standard proteins and antibodies supplied by Paraytec partners for testing. Concentrations range from 0.2 mg/ml to >40 mg/ml, a range of buffer solutions were used including PBS and samples with SDS surfactant. The samples were transferred to 300 µl glass vials and loaded onto an automated beta version of the ActiPix HT Nano-Sizing System. A summary of the standard experimental conditions is given below: System used: ActiPix HT Nano-Sizing System Capillary dimensions: Length 130 cm / detector window 1: 45 cm / detector window 2: 85 cm Programmed flow rate: 1 mm s -1 Volume of sample injected (programmable): 100 nl Detector wavelength: 214 nm Sampling Rate: 20 Hz Data filter: 0.0 s Instrumentation and Materials ActiPix TDA200 HT Nano-Sizing System (P/N TDA200 HT) ActiPix cartridge for sizing application (P/N C102SIZE360) 214 nm wavelength filter (P/N UVFILT214) 150 cm 75 x 360 μm fused silica capillary (Polymicro Technologies) Proteins & Antibodies (Paraytec Partners) Buffer solutions (Paraytec Partners) Results Figure 5. Example data report for caffeine standard 2011 Paraytec Ltd 3

4 Figure 6. Overlay of seven repeat runs of Myoglobin Figure 7. Example of a single Gaussian fit to hgh sample 2011 Paraytec Ltd 4

5 Figure 8. Example of a dual Gaussian fit for Ompf + ColN, the small distortion at the top of the main peak is likely caused by a small molecule such as a co-ion Sample Hydrodynamic Radius (nm) Caffeine 0.32 BSA 3.76 Myoglobin 2.02 hgh 2.31 IgG OmpF + coln 8.30 Maltose Binding Protein 3.24 Table 1. Representative results from different biological samples and a caffeine standard Conclusions The ActiPix TDA200 can measure hydrodynamic radii of a wide range of samples, quickly and accurately. This is achieved whilst only using very small sample volumes, across a wide concentration range. This system is fully automated and perfect for overnight analysis and quality control. References Paraytec Technical Note TN001 - Hydrodynamic radius using Taylor dispersion. Bello, M. S.; Rezzonico, R.; Righetti, P. G., Science 1994, 266, Paraytec Ltd 5

6 References Paraytec Technical Note TN001 - Hydrodynamic radius using Taylor dispersion. Bello, M. S.; Rezzonico, R.; Righetti, P. G., Science 1994, 266, Paraytec Ltd 6