Struggling with Detergents
“Detergents are widely used in protein extraction, solubilization, and denaturation,” says Lisa Bradbury, Ph.D., proteomics R&D director at Pall, “yet their presence can interfere with the performance of mass spectrometry technology.”
Given that detergents are a problem for so many downstream methods of protein analysis, a fast and efficient detergent-removal method could prove useful. Dr. Bradbury and her colleagues have evaluated the properties and behavior of SDR HyperD® resin, a mixed-mode chromatography medium. The mixed-mode properties of this resin result from a combination of a porous, spherical, silica bead with a hydrophobic polymer. The polymer is uniformly distributed throughout the silica pores allowing the specific interaction of small molecules in solution with silanol and hydrophobic groups.
SDR HyperD is effective for the removal of a family of detergents including NP-40, Triton X-100, SDS, Tween 20, and CHAPS from protein samples. Given its exquisite sensitivity to detergents, mass spectrometry can be used to detect extremely low levels of residual detergent, down to 100 ppm in protein samples.
SDR HyperD resin has a high capacity for all detergents tested, said Dr. Bradbury, and its application results in substantial improvement in MS signal intensity of proteins. Dr. Bradbury and her colleagues determined that SDR can be used in a prepacked column or with batch-mode devices to readily accommodate a variety of experimental needs. Additionally, large proteins (> 60 kDa) cannot penetrate the pores of this resin, so protein losses are minimal. Dr. Bradbury cautioned that for moderately sized proteins in the 20 to 60 kDa range, the potential for protein loss is dependant on individual protein characteristics and sample composition, and thus should be determined on a case-by-case basis.
“We found that the SDR HyperD resin provides a means for efficient detergent removal,” Dr. Bradbury stated, “especially when used in combination with the Nanosep spin devices and AcroPrep 96-well filter plates, formats that are readily amenable for high-throughput applications.”
The analysis of metabolomic data using comprehensive 2-D gas chromatography with time-of-flight mass spectrometry (GCxGC-TOF-MS) was discussed by John Heim, applications chemist at LECO. His investigations illustrate how metabolomics can be strengthened through the addition of 2-D gas chromatography to the MS platform.
“Metabolomics presents challenges that, historically, have relied heavily upon standard quadropole GC/MS, utilizing targeted methods of selected ion monitoring and tandem GC/MS/MS mass spectrometric techniques,” Heim stated. Two dimensional gas chromatography provides the resolution needed for the characterization of the small metabolite profiles.
The complex nature of metabolomic samples demands analytical solutions and instrumental methods that will identify the small molecule metabolomic profile completely as well as discover significant key components of interest. In these studies, Heim and his colleagues investigated urine samples collected from diabetic and nondiabetic humans.
Comprehensive 2-D gas chromatography expands the peak capacity of the chromatographic separation, thereby, increasing resolution and analyte characterization necessary for complex biological samples. The high- data density and narrow-peak widths inherent to GCxGC analysis require a detection system able to characterize the peak shape and small molecule metabolite identification.
The group focused on the demonstration of the benefits of the system, in that TOF-MS provides the acquisition speed necessary for optimum characterization of the complex GCxGC separations. The studies were further aimed at evaluating the statistical powers of the Chroma TOF™ software for multivariate analysis of data sets representing diseased and nondiseased states. The statistical program allows the user to recognize previously unknown chemical differences between complex samples.
The use of this software could be significant in resolving problems in metabolome screening, in which confirmation of data may be problematic, even within the same laboratory. “This establishes a viable strategy identifying significant metabolic variation in complex biological samples from diseased and nondiseased states,” Heim concluded.