Higher order structure
Vibrational spectroscopy has long been recognized as a powerful tool in the study of protein and peptide substructure. The amide I band (1700 – 1600 cm-1) probes the C=O stretch vibration of the peptide linkages which constitute the backbone structure of the protein. The differing pattern of hydrogen bonding, dipole−dipole interactions, and the geometric orientations in the α-helices, β-sheets, turns, and random coil structures induce different absorption features in the amide I band that are well correlated with these second order structures. An analysis of the absorption spectrum can be used to quantitatively determine the relative amounts of these substructures which can then be used as a powerful probe for protein characterization, chemical and thermal stability, and protein aggregation.
Despite the power of the analytical technique, measurement capabilities are typically limited to concentrations above 5-10 mg/mL for Fourier Transform Infrared Spectroscopy (FTIR) and 30 mg/mL for Raman. Ultraviolet Circular Dichroism (UV-CD), currently one of the more prevalent tools for secondary structure analysis, is relatively insensitive to beta sheet formation and has difficulty detecting the very important intermolecular beta-sheet structures which form during aggregation. While UV-CD operates at a lower concentration range than FTIR (typically ~ 0.2 – 2 mg/mL versus ~ 10 – 200 mg/mL) , it is not capable of directly measuring the higher concentration ranges typically encountered in formulation. The RedShiftBio analyzer, however, is capable of measuring protein structure over a very wide dynamic range, from 0.1 mg/mL to over 200 mg/mL. This avoids the need for sample preparation steps such as dilution or pre-concentration which may introduce variability across samples, thus requiring sample replicates and multiple measurements. Perhaps even more importantly, the analyzer enables measurement of the protein at the actual concentration of interest, whether in discovery or formulation. These are capabilities not found in today’s protein measurement tools.
The determination of protein secondary structure for alpha-chymotrypsin was performed on proteins using an automated fitting method of analysis. Figure 1 shows the analysis of alpha-chymotrypsin with good reproducibility over a concentration range from about 0.1 to 10 mg/mL.
While most technologies are restricted to performing protein analysis at concentration ranges of about one order of magnitude, measurements taken using RedShiftBio’s MMS analyzer can be performed at concentration ranges spanning more than three orders of magnitude. Figure 3 shows an example of the analysis from measurements of bovine serum albumin at concentrations ranging from 0.1 to 200 mg/mL resulting in five secondary structure components useful in protein fingerprinting.
1 Elliott A, Ambrose EJ. Structure of synthetic polypeptides. Nature 1950, 165: 921−922.
2 Fabian, H. Man̈tele, W. Handbook of Vibrational Spectroscopy; Chalmers, J. M., Griffiths, P. R., Eds.; John Wiley & Sons, Ltd: Chichester, 2002; pp 3399−3425.
3 Koenig, J. K., & Tabb, D. L. (1980) in Analytical Applications of FT-IR to Molecular and Biological Systems (Durig, J. R., Ed.) pp 241-255, D. Reidel, Boston.
4 Aichun Dong, Ping Huang, and Winslow S . Caughey. 1990. Protein Secondary Structures in Water from Second-Derivative Amide I Infrared Spectra. Biochemistry 29, 3303-3308.
5 Pots et al., 1998a Pots AM, de Jongh HHJ, Gruppen H, et al. Heat-induced conformational changes of patatin, the major potato tuber protein. Eur J Biochem 1998 ; 252 : 66-72.
6 Shivu B, Seshadri S, Li J, Oberg KA, Uversky VN, Fink AL. Distinct β-sheet structure in protein aggregates determined by ATR-FTIR spectroscopy. Biochemistry. 2013 Aug 6;52(31
7 Wei Wang, Christopher J. Roberts Aggregation of Therapeutic Proteins Wiley, Aug 30, 2010.
8 Sharon M. Kelly* and Nicholas C. Price The Use of Circular Dichroism in the Investigation of Protein Structure and Function Current Protein and Peptide Science, 2000, 1, 349-384 mg/mL. These results demonstrate good accuracy, agreeing with FTIR methods as well as published values from X-ray and UV-CD. mg/mL. Elliott A, Ambrose EJ. Structure of synthetic polypeptides. Nature 1950, 165: 921−922.
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Application Note on Microfluidic Modulation Spectroscopy
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