IR-based protein and peptide quantitation relies on the vibrations characteristic for the amide bond, a chemical structure that is not uniquely reserved to protein and peptides only. Amides are present in other biomolecules, such as nucleic acids. Therefore, pure DNA or RNA can be quantified using the amide I region of the IR spectrum.
Interestingly, the amide vibration characteristic to nucleic acids is much weaker than the one observed for proteins. Additionally, amounts of nucleic acids present in most lysates are below the instrument’s detection level, allowing accurate protein quantitation.
Some commonly used buffers (e.g., urea) or buffer components contain amides, making these sample components incompatible with IR-based detection. At the same time, IR spectra deliver much more information about the analyzed sample than just the amide signal. As discussed, one key advantage of the Direct Detect system is that it accurately quantifies proteins in the presence of reducing agents and detergents, delivering spectral information on the type of additive present in the sample.
For example, Figure 3 shows IR bands characteristic for SDS: a strong signal between 2,800 and 3,000 cm-1 contributed by vibrations of the aliphatic groups (CH2 and CH3) and a weaker band at around 1,725 cm-1 assigned to S=O bond. Similar information can be collected for other buffer components, leading to robust databases that would allow identification of unknown buffer additives.
In conclusion, the Direct Detect IR-based quantification system provides a new way of accurately quantifying proteins and peptides based on their intrinsic amide bonds. The technology provides a highly reproducible, direct, assay-free measurement that is compatible with cell lysates and many common detergents and reducing agents. The spectra can also provide additional useful information about the sample composition.