The Direct DetectTM Biomolecular Quantitation System

The Direct DetectTM system, an infrared (IR)-based spectrometry system, represents an innovation in biomolecular quantitation. The key to this advance lies in a new membrane technology for preparing and presenting aqueous biological samples to make them compatible with infrared analysis. Built upon EMD Millipore’s extensive experience in membrane technology, it employs a hydrophilic polytetrafluoroethylene (PTFE) membrane that is designed to be transparent in most of the infrared spectral region and enables application of biomolecule solutions directly onto the membrane. The Direct DetectTM system has been optimized for detection and quantitation of proteins. By measuring amide bonds in protein chains, the system accurately determines an intrinsic component of every protein without relying on amino acid composition, dye binding properties or redox potential. Introduction Limitations of traditional methods for protein quantitation. Classical methods for protein quantitation rely on colorimetric assays, such as those involving protein-copper chelation (bicinchinonic acid (BCA) and Lowry assays) and dye-binding based detection (Bradford and “660 Assay”) or ultraviolet/visible (UV-Vis) spectroscopy. Each of these methods has limitations. In colorimetric assays, protein concentration is determined by comparing signals from samples of unknown composition to signals from reference standards (composed of common proteins such as Bovine Serum Albumin (BSA)) which are prepared for every measurement. Standard curve determinations differ considerably from assay to assay, affecting reproducibility of protein concentration estimations. The UV based method relies on absorbance at 280 nm by a protein’s aromatic amino acids, predominantly tryptophan with minor contributions from tyrosine. Therefore, protein extinction coefficients can vary widely (greater than two-fold difference between extinction coefficients of albumin and immunoglobulin G). Those proteins that do not contain tryptophan, such as Protein A, cannot be quantified based on 280 nm absorbance. Amino acid analysis delivers possibly the most accurate protein quantitation; however, it is expensive and slow if samples are sent to a third party for analysis. If amino acid analysis is performed in-house, it requires time-consuming sample manipulation and specialized equipment. The Direct DetectTM system provides more universally applicable, less limited and faster protein quantitation that requires minimal amounts of often precious samples. Thanks to its new membrane technology for sample handling and presentation, the Direct DetectTM method is convenient, requires minimal sample preparation and can be used under diverse sample conditions. EMD Millipore is a division of Merck KGaA, Darmstadt, Germany How IR spectroscopy provides universal, accurate quantitation. IR spectroscopy is one of the oldest and most well established experimental techniques for the analysis of protein structure1. IR spectroscopy exploits the fact that molecules absorb radiation at specific frequencies characteristic of their structure. Designation Approximate Frequency (cm−1) Description Amide A 3,300 NH stretching Amide B 3,100 NH stretching Amide I 1,600 − 1,690 C=O stretching Amide II 1,480 − 1,575 CN stretching, NH bending Amide III 1,229 − 1,301 CN stretching, NH bending Amide IV 625 − 767 OCN bending Amide V 640 − 800 Out-of-plane NH bending Amide VI 537 − 606 Out-of-plane C=O bending Amide VII 200 Skeletal torsion Table 1. Characteristic infrared bands of the peptide bond. O O O N Cα C N Cα C N Cα C H H H