NEW YORK (GenomeWeb) – The lab of Scripps Research Institute researcher John Yates III has completed a top-down characterization of the proteome of Pyrococcus furiosus, an extremophilic species of Archaea.
Detailed in a paper published last month in Analytical Chemistry, the study combined sheathless capillary electrophoresis with mass spec on a Thermo Fisher Scientific Orbitrap Elite instrument to enable a rapid, sensitive top-down workflow.
Using the method, the researchers were able to identify 134 proteins and 291 proteoforms from P. furiosus cell lysate using a total sample amount of 270 ng and 120 minutes of total analysis time.
In an email to ProteoMonitor, Yates noted that his lab is "very interested in top-down" but has been waiting for technology – mass spec technology in particular – to improve. He added that "a number of technologies are coming together now to make it feasible to answer biological questions using top-down."
One key has been development of Thermo Fisher's Orbitrap technology, said Scripps researcher Xuemei Han, first author on the Analytical Chemistry paper. These instruments have provided scientists with a user-friendly alternative to the FT-ICR mass specs traditionally used for top-down work, she told ProteoMonitor.
In particular, the Orbitrap Elite has proven a favorite for top-down research. In 2011, for instance, Northwestern University Neil Kelleher used this instrument to identify more than 3,000 protein species in a top-down analysis of HeLa S3 cells that marked one of the first demonstrations of the feasibility of large-scale, discovery-style intact protein work.
In a 2013 paper in Molecular & Cellular Proteomics, Kelleher's lab used the Elite for the largest top-down proteomics study of a human cell line to date, identifying 1,220 proteins and more than 5,000 proteoforms in H1299 cells.
In an interview with ProteoMonitor following publication of the 2011 study, Kelleher noted that more important than the mass spec technology used was the upfront separations workflow he and his team devised – a system consisting of an initial stage of solution isoelectric focusing, followed by gel-eluted liquid fraction entrapment electrophoresis, and then nano-LC.
The upfront separation method is also key to Yates' Analytical Chemistry study, though in the case of this recent work the researchers aimed to optimize their analysis for speed and sensitivity.
To achieve this, the Scripps team turned to capillary zone electrophoresis (CZE), which separates proteins based on their mass-to-charge ratio. The method allows for rapid, efficient separations using a relatively low amount of sample. Also, the Analytical Chemistry authors noted, compared to capillary isoelectric focusing, which has also been employed for top-down analyses, CZE offers lower background levels.
In conventional LC separations, intact proteins will absorb to the stationary phase causing broadening of peaks, Han said. In CZE, meanwhile, capillary walls can be treated to reduce absorption of intact proteins, "so you have very nice peak shapes and it's very suitable for separating big proteins."
The method also requires considerably less sample than LC, Han said, noting that the researchers used roughly 10-fold less sample than they would have had to use with conventional LC. She added that in a forthcoming paper using top-down to analyze protein complexes, she and her colleagues managed to achieve a nearly 100-fold sensitivity increase compared to standard LC.
"So we have much less sample consumption, which I think is important to analyzing important biological questions because sometimes you just can't get enough sample to analyze," she said.
The researchers did, however, have to use reverse phase LC prior to their CE separation to divide the sample into fractions compatible with CE's limited peak capacity. Han noted that more extensive prefractionation could be used to make the workflow amenable to analysis or more complex organisms.
As the authors noted, a primary reason CE-MS remains less popular than LC-MS is the challenge of interfacing CE to an electrospray ionization source. Typically, a sheath liquid interface, in which a pumped sheath liquid provides contact between the CE and ESI, is used, but this approach leads to analyte dilution and decreased ionization efficiency, reducing sensitivity.
The Scripps team used a prototype sheathless CESI interface, which reduces analyte dilution, resulting in improved sensitivity.
The researchers identified 134 P. furiosus proteins and 291 proteoforms at a 2.7 percent proteoform-level false discovery rate and a 6 percent protein-level FDR. This, they noted, represents 9 percent of the 1,517 P. furiosus proteins identified to date via bottom-up experiments, a percent that is comparable to that achieved in large top-down bacterial studies, but with less sample used.
For instance, a 2013 study by researchers at Pacific Northwest National Laboratory used a single dimension of LC separation coupled to a Thermo Fisher Scientific LTQ Orbitrap Velos to identify roughly 10 percent of the Salmonella Typhimurium proteome. That analysis used 5 μg of sample – around 20 times the sample used in the Scripps P. furiosus analysis.
For their data analysis, the researchers used ProSightPC, a top-down analysis software developed by Kelleher and sold by Thermo Fisher, and Byonic, a protein ID program developed by Marshall Bern, vice president and co-founder of protein software firm Protein Metrics, and co-author on the Analytical Chemistry paper.
Currently, Han said, she and her colleagues are using the workflow for the top-down study of protein complexes in hopes that its improved sensitivity will allow them to apply top-down analyses.
Typically, the immunopurification mass spec techniques used in such work "have been done by bottom-up [proteomics]," she said. "Usually the protein amounts are very limited. So with the sensitivity of the CE, we're hoping that we can [tackle] those questions using top down."