Advantages of Next Generation Optima AUC over Proven ProteomeLab

Analytical ultracentrifugation (AUC) has been a powerful analytical technique in the analysis of macromolecules in solution since its introduction in the 1920s, resulting in impactful scientific discoveries1. AUC offers a matrix-free environment that enables the near-native characterization of proteins1, extracellular vesicles2, nucleic acids3, colloids4, and nanoparticles5. The Beckman Coulter ProteomeLab XL-A/I AUC instruments can measure sample parameters by monitoring UV/Vis absorbance and Raleigh interference separately in a single experiment.

Beckman Coulter’s recently launched Optima AUC features an improved optical system (Table 1), which positions the field to advance in the study of complex systems and the development of new applications (Figure 1). The company’s new instrumentation enables the Optima AUC to serve as a powerful tool to examine samples with high resolution, precision and accuracy.

Table 1. Comparison of specifications of Optima AUC and ProteomeLab

OPTICAL SYSTEMS

PROTEOMELAB XL-A/ XL-I

OPTIMA AUC

FASTEST DATA ACQUISITON RATE

ABS: 90 sec/cell

ABS: <20 sec/cell

INT: <5 sec/scan

MAX# OF WAVELENGTHS

3

20

WAVELENGTH PRECISION

+/- 3 nm

+/-0.5 nm

LOWEST RADIAL RESOLUTION

30 µm

10 µm

ABSORBANCE FLASH LAMP FREQUENCY

50 Hz

300 Hz

CCD CAMERA SPECIFICATIONS

2048 x 96 pixels

2048 x 1088 pixels

INTERFERENCE FRINGES

≥ 4 fringes/cell

≥ 10 fringes/cell

USABLE CONCENTRATION RANGES

ABS: .005 – 1.5 mg/Ml

INT: .025 – 3-4 mg/mL

ABS: .005 -1-2 mg/ mL Luteinizing Hormone

INT: .025 - ~4-5 mg/mL BSA**

Applications enabled by multi wavelength analysis

Figure 1. Applications enabled by multi wavelength analysis. Image credit: Beckman Coulter

  • Nanoparticles
    • Core size determination
    • Drug Payload
  • Quantum Dots
  • RNA Aptamers
  • Polysaccharides
  • Viral Vectors for Gene Therapy
    • Empty versus full capsids
  • Membrane Protein-Lipid Interaction
  • Protein-Protein Interactions
    • Fusion proteins (YFP, GFP, SNAP)
    • Heme Proteins
    • Flavins
    • Metalloproteins
  • Protein-Nucleic Acid interactions
    • Recombinases
    • Terminases
    • ATPases

Materials

Bovine serum albumin (BSA, cat. A7906-10G)) was bought from Sigma-Aldrich. ProteomeLab XL I (P/N 969341, 969340), Optima AUC (P/N B86437, C00708), An 60 Ti rotor (P/N 361964), torque stand (P/N 361318), and two sector sedimentation velocity analytical cells with quartz windows (P/N 392772) were from Beckman Coulter, Inc.

Methods

BSA sample preparation

BSA stock solution was made by dissolving BSA powder into PBS to an absorbance of 1.0 OD at 280nm. A Beckman-Coulter DU730 UV/Vis Spectophotometer with a 10mm path length was used to measure the OD of the samples. BSA was diluted to working concentrations of 0.4 and 0.9 OD in PBS.

Instrument settings

PBS buffer was the reference buffer matching the sample solvent. The 2-sector analytical cells were maximally loaded with 440 μl in both sample and reference sectors. The cells were aligned in the An 60 Ti rotor, and equilibrated at 20˚C for more than 1 hour. Subsequently, samples were spun at 42,000 rpm, 20˚C, 6 hours scanning at Abs280nm in continuous mode. The process above was carried out on the Beckman Optima AUC and the Beckman ProteomeLab XL-I AUC instruments using the same samples assembled in the same analytical cells shaken between runs.

Data analysis

The extracted data from the AUC controller was then imported into SEDFIT 14.7 g7 (www.analyticalultracentrifugation.com). Figure 2 shows the analysis of the data using the fitting parameters. The data was extracted into GUSSI (http://biophysics.swmed.edu/MBR/software.html) and plotted for c(s) with an s value minimum constraint at 0.5S.

SEDFIT fitting parameters

Figure 2. SEDFIT fitting parameters. Image credit: Beckman Coulter

Results and discussion

The deployment of new and improved software has made significant progress on data analysis. Today’s data analysis packages have now increased the limit of the current optical systems and a hardware update is needed to advance the tools of the scientific community6. Hence, the new Optima AUC optical specifications are poised to lead the field to great AUC innovations by circumventing data acquisition limitations.

The optical advances of the new Beckman Optima AUC over the Beckman ProteomeLab XL-1 were demonstrated by acquiring and analyzing the same BSA data sets on both instruments for sedimentation coefficient. Samples were run under identical conditions, and the data acquired was analyzed using the SEDFIT software.

Between the two instruments, the monomeric and dimeric BSA had a s20,w of 4.29-4.41 and 6.25-6.89, respectively, at low concentration. However, the additional trimer species emerging at 8.18 can only been seen clearly from the data acquired on the new Optima AUC (Figure 3). Also, in the blue trace of Figure 2, the data acquired on the ProteomeLab shows a peak shift of the dimer, indicating that this population is likely a smear of the dimer and trimer contribution at 6.89 c(s). No distinct third species is seen on the ProteomeLab. Conversely, all defined three populations are clearly observed from the data acquired on the Optima AUC. The comparison of the two data sets indicates that the new Optima AUC’s improved optical system can discern sparse populations within low concentration solutions.

Sedimentation velocity c(s) of BSA at 0.4 OD

Figure 3. Sedimentation velocity c(s) of BSA at 0.4 OD. Image credit: Beckman Coulter

Data acquired on both the instruments revealed all three oligomeric states of BSA at high BSA concentrations (Figure 4); however, the peaks were resolved at a higher signal-to-noise ratio by the Optima AUC, enabling a better distinction of the different peaks (Figure 4, red trace). In part, the improved precision and accuracy of the Optima AUC is owing to better meniscus fitting and enhanced radial resolution.

Sedimentation velocity c(s) of BSA at 0.9 OD

Figure 4. Sedimentation velocity c(s) of BSA at 0.9 OD. Image credit: Beckman Coulter

Figure 5 is a panel of the sedimentation velocity profiles and residual plots of every data set illustrating every scan. The increased frequency of the absorbance flash lamp makes the new instrument 4 to 5 times faster than the ProteomeLab. In comparison with the ProteomeLab, the Optima collected three times as many data points per scans owing to the increased radial resolution from 30 μm to 10 μm (Table 1). The most important aspect, as shown by the residual plot range, is the superior performance of the Optima AUC, producing less variation of the maximal residual and a tighter data fit.

Residual plots of (A) 0.4 OD in Optima AUC (B) 0.9 OD in Optima AUC (C) 0.4 OD in ProteomeLab (D) 0.9 OD in ProteomeLab.

Figure 5. Residual plots of (A) 0.4 OD in Optima AUC (B) 0.9 OD in Optima AUC (C) 0.4 OD in ProteomeLab (D) 0.9 OD in ProteomeLab. Image credit: Beckman Coulter

With the better precision and accuracy, tighter meniscus fitting can also be achieved with the new Optima AUC. The meniscus position is set as a fitting parameter during data analysis in SEDFIT, and in other data analysis programs.

The increased radial resolution of the experimental scans allows for the visual inspection and definition of the true meniscal position (Figure 6). Plot A displays the meniscus for the low concentration run on the Optima AUC and plot B displays the same cell run on the ProteomeLab. The meniscus position varies within a 50 μm precision on the Optima AUC in comparison to a 250 μm range on the ProteomeLab. There is an improvement in the true meniscus definition by five folds. Better meniscus fitting results in significant improvements in data fitting8 and the refinement of this parameter is owing to the improved radial resolution. The decrease in optical imaging artifacts results in well-determined macromolecular sedimentation parameters.

SEDFIT plot of overlay meniscus position of 0.4 OD cell in (A) Optima AUC and (B) ProteomeLab.

Figure 6. SEDFIT plot of overlay meniscus position of 0.4 OD cell in (A) Optima AUC and (B) ProteomeLab. Image credit: Beckman Coulter

Conclusion

AUC, a first principle technique, is ultimately, a powerful tool hampered only by the development and improvement of its optical systems. Beckman Coulter introduced the first AUC sample characterization tool and remains at the forefront in the field with the launch of the Optima AUC. This article has covered the advances of the new Optima AUC and demonstrated an improved optical system that facilitates data acquisition with higher resolution, precision, and accuracy.

Besides the advantages of the new optical system discussed in this article, the Optima AUC is the only commercially available equipment featuring the fastest acquisition rate of 20 seconds per cell, a broader range of sample concentration, and a multi-wavelength detector of up to 20 wavelengths with minimal time and great precision.

† Results generated from the softwares listed in this section are not guaranteed. Please see specific software tools for individual disclaimers.

References

  1. Howlett, Geoffrey J., Allen P. Minton, and Germán Rivas. "Analytical ultracentrifugation for the study of protein association and assembly." Current opinion in chemical biology 10.5 (2006): 430-436.
  2. Shulman S. The determination of sedimentation constants with the oil-turbine and spinco ultracentrifuges. Arch Biochem Biophys. 1953; 44: 230–240.
  3. Falabella, James B., et al. "Characterization of gold nanoparticles modified with single-stranded DNA using analytical ultracentrifugation and dynamic light scattering." Langmuir 26.15 (2010): 12740-12747.
  4. Diaz, Leosveys, Caroline Peyrot, and Kevin J. Wilkinson. "Characterization of polymeric nanomaterials using analytical ultracentrifugation." Environmental science & technology 49.12 (2015): 7302-7309.
  5. Plascencia-Villa, Germán, et al. "Analytical Characterization of Size-Dependent Properties of Larger Aqueous Gold Nanoclusters." The Journal of Physical Chemistry C 120.16 (2016): 8950-8958.
  6. Cölfen, Helmut et al. “The Open AUC Project.” European Biophysics Journal39.3 (2010): 347–359. PMC. Web. 5 Dec. 2016.
  7. Schuck P. Size distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and Lamm equation modeling. Biophys. J. (2000) 78:1606–19.
  8. Zhao, Huaying et al. “Current Methods in Sedimentation Velocity and Sedimentation Equilibrium Analytical Ultracentrifugation.” Current protocols in protein science / editorial board, John E. Coligan ... [et al.] 0 20 (2013): 10.1002/0471140864.ps2012s71. PMC. Web. 2 Dec. 2016.

Acknowledgments

Produced from materials authored by Julia P Luciano-Chadee and Chad Schwartz, Ph.D., Beckman Coulter Inc., Indianapolis.

Beckman Coulter Life Sciences - Auto-Cellular and ProteomicsAbout Beckman Coulter

Beckman Coulter develops, manufactures and markets products that simplify, automate and innovate complex biomedical tests. More than a quarter of a million Beckman Coulter instruments operate in laboratories around the world, supplying critical information for improving patient health and reducing the cost of care.


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Last updated: May 18, 2020 at 4:15 AM

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