Quantitation

Protein quantitation is critically important in biochemistry research and development labs in applications ranging from enzymatic studies to providing data for biopharmaceutical lot release.  Direct assay measurements include UV and visible absorption measurements relative to a standard using extinction coefficients or indirect measurements using dye base assays such as BCA, Lowry, and Bradford assays.  No one approach is universal due to the specific limitations of each approach, including aromatic residue dependency, chemical interferences in dye-based assays and the limited dynamic range of the spectroscopic tool.  One of the biggest limitations of these measurements comes from the spectroscopic tools themselves.  Conventional spectrometers have limited linearity primarily due to stray light and the instrument slit width (resolution) as well as detector linearity.  As such, the sample absorbance is targeted to a very limited dynamic range, typically between .1 and 1.5 au.  This limited range forces scientists to adjust either the sample concentration or the cell path length to acquire accurate protein quantitation.  Either alternative can be time consuming and problematic in its effect on the measurement.

Infrared absorption spectroscopy can be an effective tool for direct, label free protein quantitation.  It provides an advantage over UV/VIS methods as sample absorption bands in the infrared are much narrower and are not dependent on aromatic residues. As a result, the technique is more selective with less susceptibility to interferences.  In addition, since the IR method probes the carbonyl backbone of the protein and is not dependent on a UV chromophore, the variation is extinction coefficient is much smaller which can be an advantage in measuring unknown proteins.  However, IR spectroscopy has not been routinely used in situ due to its lower sensitivity, added cost, and difficulty of operation (i.e. background subtraction, water vapor interference, and narrow pathlength cells).  RedShiftBio’s MMS platform overcomes these issues by increasing sensitivity and significantly reducing the errors common to conventional spectroscopy.  MMS’s high resolution (<0.001

cm-1) and low stray light susceptibility increases the linear concentration range for the measurement by more than 2 orders of magnitude.  The differential measurement of microfluidic modulation spectroscopy and direct control over laser power also improves linearity by reducing signal dynamic range and maintaining high detector linearity throughout the measurement range.

Using MMS, one or at most only a few wavelengths need to be measured.  Figure 1 shows a plot at ~1656 cm-1 for BSA in the range from 0.1 to 200 mg/mL. With a minimum measurable concentration of less than 10 µg/mL (3 sigma, HEWL) and an upper limit of greater than 200 mg/mL, this offers a significant improvement over conventional absorbance based assays.

Figure 1. Differential absorbance at ~1656 cm-1 (BSA peak) plotted as a function of concentration at 0.1, 1, 10, and 200 mg/mL.  Note that at the high concentration (200 mg/ml).
Figure 1. Differential absorbance at ~1656 cm-1 (BSA peak) plotted as a function of concentration at 0.1, 1, 10, and 200 mg/mL.  Note that at the high concentration (200 mg/ml).

1 Noble JE, Bailey MJ., Quantitation of protein, Methods Enzymol. 2009;463:73-95. doi: 10.1016/S0076-6879(09)63008-1. Review.

2 Douglas A. Skoog , F. James Holler Stanley R. Crouch, Principles of Instrumental Analysis 6th Edition, Cengage Learning, 2006.

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