Standard methods for protein quantitation rely on colorimetric assays like those involving protein-copper chelation (BCA and Lowry assays) and dye-binding based detection (Bradford and 660 Assay) or ultraviolet (UV) spectroscopy.
While colorimetric assays are easy to use, they are highly sensitive to sample components (detergents and reducing agents), protein composition and structure, and dye-binding properties. In these assays, protein concentration is determined by comparing signals from samples of unknown concentration to signals from reference standards (composed of common proteins such as bovine serum albumin), which are prepared for every measurement. Standard curve determinations differ considerably from assay to assay, affecting measurement reproducibility.
The BCA assay involves a two-step chemical reaction requiring incubation, and delivers results that can vary due to the kinetic rate of the reaction. In addition, phospholipids, chelating agents, reducing agents, and certain nonionic, oxidizing detergents can negatively impact the assay signal. The Bradford assay relies on binding of Coomassie Brilliant Blue G250 to basic amino acids, particularly arginine.
As a result, the assay outcome depends on the number of basic amino acid residues in the analyzed protein, which can vary greatly between proteins. Additionally, because the assay requires standard curve generation for every experiment, the proportion of basic amino acid residues in the protein standard needs to be similar to that of the protein being assayed.
UV spectroscopy-based quantification methods rely on the absorbance at 280 nm by a protein’s aromatic amino acids, predominantly tryptophan and tyrosine. Therefore, the absorbance at 280 nm of different proteins can vary widely (greater than twofold difference between extinction coefficients of albumin and immunoglobulin G). Additionally, proteins that do not contain aromatic amino acids, such as Protein A, cannot be quantified based on 280 nm absorbance.
Amino acid analysis delivers possibly the most accurate protein quantitation. The procedure consists of several steps—hydrolysis, derivatization, separation, and detection followed by quantitation. The results can be greatly influenced by variability in the inherent liability of a given sequence to hydrolysis, derivatization conditions, or sample contaminants (mainly presence of nonvolatile amines like Tris or glycine). Additionally, this method is expensive and may take time to obtain results if samples are sent to a third party for analysis.
Traditional Peptide Quantitation
Classical methods for peptide quantitation rely on the weight of the lyophilized powder, UV spectroscopy, or amino acid analysis.
Establishment of actual peptide concentration based on the weight of the lyophilized powder is inaccurate because the analyzed powder can contain a significant quantity (10−70%) of bound water, salts, or counterions. One UV spectroscopy-based peptide quantitation method relies on absorbance at 280 nm. This wavelength can be used to estimate peptide concentration if tryptophan and/or tyrosine residues are present in the sequence. Therefore, peptides that do not contain aromatic amino acids cannot be quantified using this method.
It is possible to determine peptide concentration by measuring absorbance at 205 nm. However, this measurement is far more prone to external influence, since many solvents and other chemicals will absorb at this wavelength. A high-quality, dual-beam spectrophotometer is required in order to reduce the effects of nonspecific absorption and to measure low concentrations.
The Direct Detect™ spectrometer (EMD Millipore), an infrared (IR)-based protein quantitation system, represents an innovation in biomolecule quantitation. The key to this advance lies in the membrane technology for preparing and presenting aqueous biological samples to make them compatible with infrared analysis. The technology employs a hydrophilic polytetrafluoroethylene (PTFE) membrane that is spectrally “transparent” in the amide I and II regions used for protein and peptide detection and quantitation, enabling application of biomolecule solutions directly onto the membrane without added sample preparation.
By measuring amide bonds in protein and peptide chains, the system accurately determines an intrinsic component of every protein and peptide without relying on amino acid composition, dye binding properties, reaction kinetics, or redox potentials.
IR spectroscopy exploits the fact that molecules absorb radiation at specific frequencies characteristic of their structure and functional groups. Amide bonds within the protein absorb radiation in multiple regions of the IR spectrum, including a strong band at 1,600−1,690 cm-1 (amide I). In order to determine protein concentration, the Direct Detect system uses the intensity of the amide I band, which is assigned to C=O stretching vibration of the peptide bond (about 80%) with a minor contribution from C-N stretching vibration (about 20%), as shown in Figure 1. A key advantage to this system is that IR-based amide bond quantitation is not subject to interference from detergents, reducing agents, and chelators.