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Below is a description of how Fourier Transform Infrared (FTIR) spectroscopy enables protein quantitation with minimal dependence on protein sequence.
- Basic Principles of Infrared Spectroscopy
- Applications of Mid-Infrared (MIR) Spectroscopy in Protein Analysis
- Principles of Quantitation Using the Direct Detect® FTIR Spectrometer
Protein quantitation in presence of lipids within a complex cell lysate is possible because the most intense regions of lipid absorbance are spectrally distinct from the protein’s Amide I and Amide II signals
Basic Principles of Infrared Spectroscopy
Infrared Spectroscopy (IR) is the analysis of infrared light interacting with a molecule. IR spectroscopy exploits the fact that molecules absorb radiation at specific frequencies characteristic of their structure.Addition of the Fourier Transform, in the 1960s, to IR analysis resulted in higher resolution and decreased noise, increasing the attractiveness of this technique. The infrared portion of the electromagnetic spectrum is divided into three regions: the near-, mid- and far-infrared, named for their relation to the visible spectrum.
Mid-infrared (MIR) spectroscopy is based on the absorption of radiation in the approximate range 4000–400 cm-1, and is considered among the most promising spectroscopic techniques for biomedical research and diagnostics. Components of complex biomolecular mixtures (proteins, lipids, carbohydrates etc.) have separable IR spectra therefore they can be analyzed simultaneously.
Applications of Mid-Infrared (MIR) Spectroscopy in Protein Analysis
MIR spectroscopy is one of the most well-established techniques for the analysis of protein and peptide structure. Several Amide bands have been identified in MIR spectroscopy, allowing for characterization of proteins. Among these, Amide I (1600 – 1690 cm-1) and Amide II (1480 – 1575 cm-1) are recognized as the most representative of all vibration modes.Attenuated total reflection (ATR) spectroscopy and transmission flow-through cells used in combination with complex chemometric data analysis have recently enabled quantitative protein analysis directly from aqueous samples. However, to enhance sensitivity, the multivariate approach (e.g. partial least-squares analysis (PLS)) is usually applied to data analysis. Because this multivariate approach requires a certain level of IR expertise for proper manipulation of spectra and data analysis, the method has not been routinely used in life science laboratories.
Principles of Quantitation Using the Direct Detect® FTIR Spectrometer
Sample analysis by the Direct Detect® FTIR spectrometer starts with a hydrophilic polytetrafluoroethylene (PTFE) membrane engineered for sample application and retention. The membrane is transparent in the MIR regions used for protein and lipid/detergent analysis.Because the method requires minimal volume (2 µL), the method can be successfully applied to the analysis of precious material available in limited quantities.
A simple univariate (Beer–Lambert) analysis, applied by Direct Detect® Spectrometer, relies on integration of Amide I band and uses directly searchable absorptions on the spectrum curve. Protein quantification by MIR, while still based on a curve-fitting technique, presents substantial advantages over other current methodologies (such as UV absorbance or colorimetric assays):
- Unlike UV absorbance at 280 nm, MIR-based protein quantitation is much less dependent upon amino acid composition.
- Amide bond quantitation by MIR is not subject to signal interference from many common biological buffer components, such as detergents, reducing agents and chelators, demonstrating superiority over standard colorimetric assays.
- In contrast to UV spectroscopy or any other known protein quantitation method, simple, MIR-based analysis can also be employed for simultaneous analysis of lipids or detergents.