In life science research, detergents are primarily used in sample preparation to liberate cellular components through membrane
disruption and to solubilize lipid-associated proteins. However, detergents can interfere with applications such as enzyme-linked
immunosorbent assays, isoelectric focusing, nuclear magnetic resonance spectroscopy, and mass spectrometry, and therefore,
often need to be removed before analysis. Here we report a novel infrared-based method that permits fast and impartial analysis
of detergent removal from biological samples. Because this method is spectrally based and label-free, determination of protein
concentration can be performed simultaneously. An additional benefit of this technique is the small volume (2 ÁL) required
for analysis, a significant fact given the often precious nature of biological samples.
Detergents, water-soluble surface-active agents distinguished by their amphipathic structure, are used extensively in protein
biochemistry. Principally, this large family of molecules can be used to isolate, solubilize, and stabilize membrane proteins
(1–5). Depending on their structural properties, certain detergents promote solubilization and disaggregation of recombinant
proteins during the process of extraction and purification (1), and others facilitate protein stabilization and crystallization
(6). Detergents are also used to reduce background by minimizing nonspecific binding or protein precipitation in a wide range
of immunoassays (2,4).
All detergent molecules contain a hydrophilic head and a hydrophobic chain (or tail). This unique composition permits their
spontaneous, but ordered aggregation in aqueous media resulting in the formation of stable micelles. In their pure form, detergent
micelles contain from three to several hundred molecules, and the number of molecules in the micellar form (aggregation number)
is an important characteristic of the detergent. Consequently, detergent micelles can vary greatly in size and the molecular
weight of the micelle represents another useful parameter describing detergent properties. Micelle formation is initiated
after a detergent content reaches a certain threshold termed the critical micelle concentration (CMC). Generally, detergents must be used at concentrations above their CMC to be effective (1–4).
The extraction of proteins from cells or tissue requires detergents to facilitate dissociation and solubilization. Although
detergents are critical to this process, as well as numerous other preparative methodologies used in protein research, they
can interfere with many downstream applications, thus limiting analytical sensitivity, so they have to be removed before the
analysis (7–9). The most commonly used detergent removal methods include size-based exclusion (dialysis and gel filtration),
hydrophobic adsorption, and ion-exchange chromatography (10). The choice of an appropriate method is dictated by inherent
properties of the detergents used including hydrophobicity, CMC, micelle size, and charge.
UV absorbance, an array of colorimetric methods, and mass spectrometry (MS) are current options to monitor and optimize the
efficiency of detergent removal (8,11,12). Because of the presence of aromatic rings, the concentration of compounds, such
as Triton X (100 and 114) and NP-40, can be estimated by absorbance at 275 and 280 nm (8). However, application of this method
is very limited given that detergent's absorbance overlaps with that of the protein. Specifically, for complex biological
samples, such as cell culture or tissue lysates, it is impossible to establish pre-existing levels of protein and distinguish
the absorbance signals produced by detergent and protein. Total organic carbon (TOC) analysis, yet another method applied
to monitoring detergent removal, is also confounded by protein interference and is thus restricted to measuring detergent
levels in the flow-through (removed) fraction. The use of liquid chromatography–tandem mass spectrometry (LC–MS) or matrix-assisted
laser desorption–ionization (MALDI) has been reported in the case of monitoring the removal of Tween 20 and BRIJ-35 (8). SDS
concentration is typically measured by a colorimetric approach using Stain-All dye (11). Similarly, concentrated sulfuric
acid and phenol (12) are frequently used for concentration estimations of glycosidic and bile salt-based detergents like octyl
glucoside and CHAPS. In summary, none of the existing platforms or methodologies appears capable of broad-based detergent
content measurement in biological samples.
Here, we report on the development of a novel Fourier transform infrared (FT-IR)-based method for fast and impartial analysis
of detergent concentration in biological samples. The method uses a hydrophilic polytetrafluoroethylene (PTFE) membrane engineered
for sample retention and optimal transparency in regions of the IR spectra used for analysis of biological samples. Aqueous
samples are applied directly onto the membrane, dried, and analyzed by FT-IR, with minimal volume requirement (2 ÁL). Because
of the specificity of the absorbance bands, the technique offers simultaneous detection of multiple species, such as protein
and detergents reported here, without interfering with one another. This dual capability is well suited for the optimization
of protein preparative processes where the reduction of detergent content is ultimately required.