Standard Curve Generation
Assay-free, IR-based protein and peptide quantitation involves measuring intensity of the amide I band in the protein/peptide spectrum and subtracting the signal contributed by the buffer alone. The resulting amide I signal strength is then converted to the concentration by comparing it to the standard curve. The instrument requires a standard curve to be generated only once. Results obtained on 21 systems preloaded with the same standard curve showed highly reproducible protein quantitation (4.23% CV; data not shown). Benchmarking every experiment to the same, robust standard curve provides more reproducible results and facilitates intra-assay comparison across multiple experiments.
Dynamic Range, Accuracy, and Reproducibility
The system has been optimized for protein and peptide quantitation within the range 0.25−5 mg/mL. The accuracy of concentration estimation within the instrument dynamic range was established using single protein solutions, protein mixtures, and peptide solutions. Resulting concentration determinations were comparable to the data from amino acid analysis.
In contrast to other currently available methods estimating protein concentration, the Direct Detect system allows the user to perform multiple measurements without additional sample requirement. The assay-free sample card permits multiple analyses of the same sample that can be separated by hours or days.
Repeated measurements of the assay-free card show as little as 1.5% error on a single instrument and 2.6% error across multiple units (data not shown).
The accuracy of quantitation is not affected by the presence of reducing agents and detergents. Figure 2 compares performance of colorimetric assays and IR-based protein quantification in presence of additives. While concentration determination obtained on the Direct Detect system matches the standards regardless of measurement conditions (A), the Coomassie Plus (Bradford) assay results are affected by the presence of 1% sodium dodecyl sulfate (SDS) (B), and the Micro BCA™ assay could not provide any reliable concentration determination in the presence of 50 mM dithiothreitol (DTT) (C).
A sample spectrum (Figure 3) shows that SDS does not have a strong signal in the amide I region and explains why SDS does not interfere with IR-based protein quantitation. Similarly, the IR spectra of reducing agents such as DTT or β-mercaptoethanol do not interfere with amide I quantitation (spectra not shown).
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.