Andrew Chambers and colleagues from University of North Carolina, Chapel Hill, described an integrated microfluidic capillary electrophoresis-MS method using dataindependent multiplexed fragmentation to perform high-throughput proteomics.
The technique could be applied to a bottom-up proteomic approach and for characterization of protein-based biotherapeutics, for example. The capillary electrophoresis device is 42 cm long, with an MS port located at one corner through which a sample is sprayed into a Q-TOF mass spectrometer.
The flow rate of the system is 18 nm/min, reported Chambers, and it is possible to deliver overlapping injections into the capillary electrophoresis device at injection intervals of three minutes. Injecting a second aliquot of the same or a different sample before the previous sample is delivered to the MS did not result in sample carryover from run to run.
Microfluidic CE-MSE of a tryptic digest using the Waters SYNAPT G2 yielded a peak capacity of about 70, with about 12 points/peak in a 40 millisecond scan time, generating sufficient data for peptide identification. The researchers compared three runs each of CE-MSE and LC-MSE. The former identified 136 peptides and the latter 141, with an overlap of 120 peptides.
“In the future we want to integrate onchip sample cleanup and preconcentration to minimize sample waste” and incorporate quantitative analysis, said Chambers.
Rawi Ramautar and colleagues from Leiden University Medical Center, in collaboration with Beckman Coulter talked about how changing levels of low molecular weight metabolites present in biological fluids may closely associate with phenotype. The large diversity and dynamic range of these metabolites makes it difficult to use a single technique for global metabolome analysis.
Ramautar's group chose capillary electrophoresis, a charge-based separation technique, because metabolites present in body fluids tend to be highly polar and ionogenic. The method is fast and efficient, requires minimal sample preparation, and incorporates in-capillary sample preconcentration, according to Ramautar.
Beckman designed its CESI (capillary electrophoresis nano-ESI)-MS system with a sheathless interface. The researchers compared urine profiling with CE-MS performed on 8-nL sample injections using either a sheathed or sheathless liquid interface. They showed that the limits of detection (LOD) were at least 10- to 30- fold better with the sheathless approach, and it yielded many more molecular features of the urinary metabolites. They reported an LOD range of 11–115 nmol and a dynamic range of two to three orders of magnitude with a sheathless interface.
“Nanoflow separation technologies are increasing the speed and precision with which we can validate biomarkers,” says Ian Pike, Ph.D., COO of Proteome Sciences. The company performs protein biomarker discovery, validation, and mass spec-based assay development and has introduced commercial assays for Alzheimer's disease.
“MS-based biomarker validation is transforming the potential for clinical applications of biomarkers,” Dr. Pike added. It is now possible to analyze a peptide panel against a plasma sample in about 20 minutes, and “we would like to reduce that to less than 10 minutes” in a format that would be amenable to on-site testing in a physician's office.
Proteome Sciences, in collaboration with Thermo Fisher Scientific, developed isobaric tagging technology in Tandem Mass Tags (TMT®) that allows users to assay up to six samples per run on the Thermo Orbitrap Velos mass spectrometry system. In addition to providing multiplexing capabilities, the technology enhances the ability to measure peptide isoforms and to differentiate posttranslational modifications.
Proteome Sciences recently announced that it was selected to participate in the new “Denamic” project to develop tools and methods to monitor the neurotoxic effects of environmental pollutants that may adversely affect cognitive skills and cause developmental disorders in children. The project will be funded by a European Union Framework 7 grant. The study will involve mass spectrometric analysis of TMT-labeled peptides, which will form the basis for the development of biomarker assays.