How IR Spectroscopy for Protein Quantitation Works
Classical methods for protein quantitation rely on colorimetric assays, such as those involving protein-copper chelation (bicinchoninic acid (BCA) and Lowry assays) and dye-binding based detection (Bradford and “660 Assay”) or ultraviolet (UV) spectroscopy.
Problem: Classical methods for protein quantitation rely on colorimetric assays, such as those involving protein-copper chelation (bicinchoninic acid (BCA) and Lowry assays) and dye-binding based detection (Bradford and “660 Assay”) or ultraviolet (UV) spectroscopy. Each of these methods has limitations. In colorimetric assays, standard curve determinations differ considerably from assay to assay, affecting reproducibility of protein concentration estimations1. Similarly, the UV spectroscopy-based method relies on absorbance at 280 nm by a protein’s aromatic amino acids, predominantly tryptophan and tyrosine. Therefore, those proteins, such as Protein A, that do not contain aromatic amino acids, cannot be quantified based on 280 nm absorbance. Amino acid analysis delivers possibly the most accurate protein quantitation; however, it is expensive and slow if samples are sent to a third party for analysis. If amino acid analysis is performed in-house, it requires time-consuming sample manipulation and specialized equipment. In all of these methods, sensitivity to buffer components and contaminating biomolecules can often render the assay unreliable2.
Solution: The Direct Detect™ system (EMD Millipore), an infrared (IR)-based spectrometry system, represents an innovation in biomolecular quantitation. The key to this advance lies in a new membrane technology for preparing and presenting aqueous biological samples to make them compatible with infrared analysis. It employs a hydrophilic polytetrafluoroethylene (PTFE) membrane that is designed to be transparent in most of the infrared spectral region and enables application of biomolecule solutions directly onto the membrane. The system has been optimized for detection and quantitation of proteins. By measuring amide bonds in protein chains, the system accurately determines an intrinsic component of every protein without relying on amino acid composition, dye binding properties or redox potential.
IR-based protein quantitation using the Direct Detect™ system involves measuring the intensity of the Amide I signal in the protein’s IR spectrum, and subtracting the signal contributed by buffer alone in that region. A sample spectrum (Figure 1) shows that the IR spectrum of SDS does not have a strong signal in the Amide I region that would significantly interfere with protein quantitation. As a result, IR-based quantitation retains accuracy and reproducibility in the presence of SDS. Similarly, the IR spectra of reducing agents such as DTT do not interfere with Amide I quantitation (spectra not shown). Also, IR-based quantitation of proteins is independent of time. Unlike colorimetric Bradford and micro BCA assays, which are based on indirect detection of a secondary reaction and whose signals continue to change over time, the IR signal of a protein is not affected by the time between sample preparation and data acquisition.
The stability of the IR signal over time, together with compatibility with detergents and reducing agents, make IR-based quantitation a more convenient, flexible, universal approach to measuring protein levels in complex mixtures, compared to the Bradford and BCA colorimetric assays. Also because assay-free, IR-based quantitation only requires a standard curve to be generated once (instead of for every experiment), quantitating single samples using the Direct Detect™ system is faster than quantitating single samples using colorimetric assays. Benchmarking every experiment to the same, robust standard curve also provides more reproducible results and facilitates intra-assay comparisons across multiple experiments referencing the same standard curve.
For more information on the Direct Detect™ system, please visit http://www.millipore.com/directdetect
The characteristic peaks of the SDS IR spectrum are distinct from the Amide I region of the protein spectrum.