In January of 2013, members of the American Society for Mass Spectrometry (ASMS) met at the Sanibel Conference, St. Pete Beach, Florida, to discuss top-down mass spectrometry (MS) based proteomics. Researchers aim to increase the throughput of top-down proteomics while still preserving the sensitivity and viability of this approach. Collectively, researchers predict that top-down proteomics will, one day, be used to define every human measurement.1
Much of the success of top-down proteomics strategies results from the development of the high resolution Orbitrap family of mass spectrometers. During the meeting, Mike Senko (Thermo Scientific) spoke about the direction of technology for these high resolution machines. Instruments of the future will require a specialized preamplifier for rapidly detecting the first “beat” of the transient, Senko told the meeting’s attendees; mass spectrometers also will require a careful cooling of ions in the higher-energy C-trap dissociation (HCD) chamber of the mass spectrometer. Another expansion benefiting top-down proteomics is an increased capacity for handling molecules with higher molecular weights. The triple quadrupole–Orbitrap hybrid, Q-Exactive mass spectrometer (Thermo Scientific), for example, contains modification of ion optics HCD chamber pressure and an extension of masses beyond 10,000 m/z.1
Advanced instrumentation is vital to sensitive research, such as the study of intact native antibodies, as demonstrated at the ASMS conference. Research by Rosati et al. published in November 2012 explored a top-down strategy to analyze native antibodies using Orbitrap technology (Thermo Scientific).2 A mixture of ten monoclonal antibodies were deglycosylated and analyzed via MS. Results revealed that nine distinct signals could be clearly observed, which could be assigned to nine out of the ten antibodies. The antibody that could not be distinguished was below the threshold for isotopic distribution at 20 Da and would have needed 25 Da or more separation to be distinguishable. The Rosati group was also able to detect individual components present in mixtures of glycosylated mAbs and the relative amounts of each component.
As the concluding speaker of the ASMS meeting, Neil Kelleher described his suggestions for a cell-based, top-down human proteome project.3 Historically, the majority of research, including work from the Human Proteome Organisation (HUPO), has focused on a “bottom-up” strategy. Kelleher patterns his ideas after the Human Genome Project; those ideas include goals of analyzing 4,000 cell lines and characterizing 1 billion proteoforms present in all cell types and body fluids by the year 2030. The cell-based project relies on, first, the understanding of cellular conditions that produce proteins. This project is also designed to complement the Chromosome-centric Human Proteome Project (C-HPP) currently underway. Although this proposed strategy is still in the planning stages, Kelleher seems confident his work will bring new insights into biological systems and ultimately lead to the discovery of improved drug targets.
1. Whitelegge, J. (2013) “Intact protein mass spectrometry and top-down proteomics,” Expert Review of Proteomics, 10(2) (pp. 127–129).
2. Rosati, S., et al. (2012) “Exploring an Orbitrap analyzer for the characterization of intact antibodies by native mass spectrometry,” Angewandte Chemie International Edition in English, 51(52) (pp. 12992–12996).
3. Kelleher, N.L. (2012) “A cell-based approach to the human proteome project,” Journal of the American Society for Mass Spectrometry, 23(10) (pp. 1617–1624).