Characterization of the proteome
Protein-level biology holds the answers to some of the most pressing health questions on researchers’ minds. From identifying important disease biomarkers to aiding drug development by mapping protein interactions, much can be gained from studying the processes within the proteome. But how do we use this enormous wealth of knowledge to further advance science?
Understanding the processes in the proteome requires the characterization and analysis of the proteins contained. This is not an easy task. The challenges surrounding this protein analysis can be boiled down to two complicating factors: scale and complexity. First, the proteome can contain hundreds of proteins at different concentrations, which requires techniques with large dynamic measurement ranges. Second, proteins exist not just as a single species, but as a variety of proteoforms. These proteoforms are extremely difficult to characterize because of their subtle differences – yet this characterization is extremely important because proteoforms carry different biological functions that need to be established. Accurate and high-resolution analysis techniques are absolutely necessary to unravel the complexity of the proteome and enable better biotherapeutic analysis and design.
A breakthrough in the analysis of complex samples
The traditional tool of choice for characterizing large heterogeneous protein complexes is mass spectrometry (MS). MS measures the mass to charge ratio (m/z) of protein ions and provides high-resolution measurements that can address the complexity of proteoforms. In particular, native MS is an advantageous tool that can characterize the composition and stoichiometry of large heterogeneous protein complexes in their native state.
Despite being an extremely useful technique, current MS approaches for the analysis of large or complex macromolecules still face major challenges. Most importantly, there can be an overlap in charge state and isotopic distributions caused by residual solvation, ionic adducts, and post-translational modifications. This overlap leads to a high level of complexity that is unresolved for larger complexes – around 150 kDa – and leads to spectra that are difficult to handle m/z-based unfolding. Now, new developments in MS technology could provide the answer to analyzing previously unsolvable targets.
Find clarity with Direct Mass Technology mode
Charge detection mass spectrometry (CDMS) is emerging as a solution to address the challenges of characterizing complex proteoforms by providing data for larger and more complex macromolecules directly in the mass domain. CDMS can be done concurrently with the traditional one m/z Measurements using the Thermo Scientific™ Orbitrap™ Analyzer, which adds charge detection capabilities to an existing, established system. This approach provides parallel individual ion measurements leading to a direct and accurate mass determination without the need for it w/z-based deconvolution from charge-state or isotope-resolved signals in native ensemble measurements.
CDMS offers a variety of advantages for the analysis of large protein complexes. One of the most important advantages is that the resolution is increased by up to 20 times compared to traditional ensemble ion measurements. In addition, it not only offers an increased dynamic range for better protein detection, but also raises the upper limit of accessible mass measurements into the megadalton range. CDMS also requires less sample or a less concentrated sample due to its extraordinary sensitivity to achieve excellent results compared to previous approaches.
So how does it work? The sample is swept, collecting hundreds of individual ions in parallel per spectrum (Figure 1). That m/z is determined by the frequency of the axial oscillation and the Thermo Scientific™ Direct Mass Technology™ mode additionally calculates the integrated induced intensity signal over time and outputs a Diagram of selective temporal overview of resonant ions (STORI).. The slope of this plot of a single ion is proportional to its charge, allowing the determination of e.g. From multiplying m/z and e.gthe mass of each individual ion can be calculated to obtain a high resolution mass range spectrum ready for analysis.
illustration 1: Scheme showing how high resolution mass domain spectra are obtained via CDMS.
A tool that matches the biological complexity
Researchers are studying the potential of CDMS for analyzing complex solutions of larger macromolecules. And the results are incredible. A study analyzed the potential of CDMS to measure proteoforms and their complexes in both denatured and native modes of operation. Over 500 proteoforms were detected by CDMS – standard ensemble MS alone did not detect any (Figure 2). Another study used CDMS to examine intact proteoforms. Low frequency fragment ions containing many hundreds of previously undetectable residues were identified, showing a 48% increase in achievable sequence coverage compared to conventional readout for a 40 kDa protein. Without this technology, researchers would not have detailed insight and would remain in the dark about the existence of these large species.
Complex biotherapeutic characterization can sometimes be achieved by traditional ensemble MS methods, but often requires multiple experiments or extensive sample processing. A recent study examining etanercept, a tumor necrosis factor (TNF) inhibitor, identified the coexistence of over 100 glycoforms using native MS and a Thermo Scientific™ Q Exactive™ Ultra-High Mass Range (UHMR) mass spectrometer. By using Direct Mass Technology mode instead of ensemble measurements, more reliable characterization can be achieved to reveal the complex heterogeneity present in unit resolution biotherapeutics (Figure 2).
figure 2: Analysis of the native etanercept FC fusion protein (approximately 125 kDa monomer) using a Q Exactive UHMR mass spectrometer with Direct Mass Technology mode (top) and traditional ensemble MS (bottom). The Direct Mass Technology mode shows a much broader proteoform distribution compared to the traditional approach.
CDMS breaks records for the size of species it can detect. In fact, CDMS has given unprecedented isotopes Resolution for species with masses in the hundreds of kDa with an Orbitrap instrument with Direct Mass Technology mode. While much research has been done with CDMS, now that the technique is commercially available, it has much to offer laboratories studying complex macromolecules.
Unlocking the potential of the proteome
Proteomes contain a vast amount of information waiting to be used. CDMS gives us the opportunity to understand proteoforms in unprecedented detail and make the most of these valuable insights. Now the immense complexity of biology can be explored with unprecedented clarity, even observing low-frequency events. Not only is CDMS an important tool for proteomic analysis, but its capabilities are also ideal for application in complex biotherapeutic analyses. The ability to improve our understanding of complex analytes will ultimately guide researchers and enable the development of better next-generation biotherapeutics and drug modalities for improved healthcare.
About the author:
Daniel Hermanson began his research career in the field of chemical biology, focusing on the use of mass spectrometry to understand biological processes. He received his PhD from Vanderbilt University in Nashville, TN, studying substrate-selective inhibition of cyclooxygenase-2. He then did postdoctoral research at the Scripps Research Institute, applying activity-based protein profiling to study the function of unannotated proteins. Daniel joined Thermo Fisher Scientific as a Field Applications Scientist supporting mass spectrometry customers with instrument and applications training. He is currently Senior Product Marketing Manager and focuses on advanced mass spectrometry products and software.