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 μL) 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 μL). 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.