Higher order structure

Microfluidic Modulation Spectroscopy is a powerful tool in the study of protein and peptide higher order structure. The amide I band (1700 – 1600 cm-1) probes the C=O stretch vibration of the peptide linkages which constitute the backbone of the protein structure.

Alpha Helix and Beta Sheets

The differing pattern of hydrogen bonding, dipole−dipole interactions, and the geometric orientations in the alpha helices,   beta sheets, turns, and random coil structures induce different absorption features in the amide I band. These are well correlated with higher order structures. An analysis of the absorption spectrum can be used to quantitatively determine the relative amounts of the substructures. These can then be used as a powerful probe for protein characterization, chemical and thermal stability, and protein aggregation.

Shortcomings of FTIR, Raman and Circular Dichroism for Protein Secondary Structure Analysis

Despite the power of vibrational spectroscopy, 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 higher order structure analysis, is relatively insensitive to beta sheet formation. It also has difficulty detecting the very important intermolecular beta-sheet structures which form during protein aggregation.  While circular dichroism 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 AQS3pro, however, is capable of measuring protein secondary 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.

Higher Order Structure Data Using the AQS3pro

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 AQS3pro 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.

Related Resources:

Protein Secondary Structure Changes in Amyloid Formation

  • Poster: Early Events in Amyloid Formation by Lysozyme Detected by Microfluidic Modulation Spectroscopy

Analytical Characterization of Secondary Structure of Monoclonal Antibodies

  • Application Note: Monoclonal Antibody Analysis by Microfluidic Modulation Spectroscopy in a Complex Formulation Buffer (1 to 150 mg/mL)
  • Poster: Microfluidic Modulation Spectroscopy Analysis of a Monoclonal Antibody at Different Concentrations
  • Poster: Microfluidic Modulation Spectroscopy of a Biotherapeutic at Low to High Concentrations without Interference from Formulation Excipients
  • Webinar: Immunogen, Celldex and RedShiftBio Discuss Microfluidic Modulation Spectroscopy’s Ability to Monitor Changes to the Secondary Structure of Proteins
  • Webinar: Amgen and RedShiftBio Discuss how Microfluidic Modulation Spectroscopy is used to understand the secondary structure of mAb proteins
  • Poster: Microfluidic Modulation Spectroscopy (MMS) - a novel automated infrared (IR) spectroscopic tool for secondary structure analysis of biopharmaceuticals with high sensitivity and repeatability

Microfluidic Modulation Spectroscopy for Measuring Protein Secondary Structure and Similarity

  • Journal of Pharmaceutical Sciences paper: Comparing Microfluidic Modulation Spectroscopy with Circular Dichroism and FTIR Spectroscopy for Characterizing Secondary Structure of Proteins
  • Webinar: “Using Microfluidic Modulation Spectroscopy to Monitor Protein Misfolds and Structural Similarity”
  • Application note: Microfluidic Modulation Spectroscopy (MMS) Fills an Analytical Gap with a Lower LOQ for Measuring Protein Misfolds and Structural Similarity
  • Poster: HOS Study for IgG Samples Spiked with Different Amount of BSA by Microfluidic Modulation Spectroscopy

Measuring Secondary Structure of Proteins Over Wide Concentration Ranges

  • Application Note: Thermal Denaturation Analysis of Bovine Serum Albumin over Wide Concentration Range by Microfluidic Modulation Spectroscopy
  • Poster: Thermal Denaturation Analysis of Bovine Serum Albumin (BSA) by Microfluidic Modulation Spectroscopy
  • Poster: Repeatability, Concentration Linearity and High Order Structure Analysis of an IgG1 Sample by Microfluidic Modulation Spectroscopy (MMS)
  • Poster: Comparability, Similarity, Linearity and High Order Structure Analysis of an IgG1 Sample by Microfluidic Modulation Spectroscopy
Figure 1. Secondary structure for alpha-chymotrypsin measured over 2 orders of magnitude of concentration (0.1 to 10 mg/mL). Results agree with conventional FTIR results (likely Dong paper, 20 or 30mg/mL).
Figure 1. Secondary structure for alpha-chymotrypsin measured over 2 orders of magnitude of concentration (0.1 to 10 mg/mL). Results agree with conventional FTIR results (likely Dong paper, 20 or 30mg/mL).
Figure 2. Secondary structure for hen egg white lysozyme (HEWL) from 7 separate measurements of 10 mg/mL samples, taken over one month, showing standard deviation of about 1%.
Figure 2. Secondary structure for hen egg white lysozyme (HEWL) from 7 separate measurements of 10 mg/mL samples, taken over one month, showing standard deviation of about 1%.
Figure 3. RedShiftBio system measurements of protein secondary structure of Bovine Serum Albumin over a range of concentration from below 0.1 mg/mL to 200 mg/mL showing good analysis results over three orders of magnitude in concentration.
Figure 3. RedShiftBio system measurements of protein secondary structure of Bovine Serum Albumin over a range of concentration from below 0.1 mg/mL to 200 mg/mL showing good analysis results over three orders of magnitude in concentration.

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.

Enter your details below and we'll send you the Application Note on Microfluidic Modulation Spectroscopy

RedShiftBio®

RedShiftBio® is an innovative provider of analytical instrumentation for the research, development and manufacturing of protein therapeutic drugs.

Read More

Awards

TAS-footer
prism-logo-footer

Contact Us

RedShift BioAnalytics Inc.

131 Middlesex Turnpike
Burlington, MA 01803

T: 781.345.7300
F: 781-345-7301
E: info@tired-planes.mysites.io.flywheelstaging.com

RedShift™, RedShiftBio®,  See Change®, and AQS3™ are trademarks of RedShift BioAnalytics, Inc.