Revolutionizing Biomolecule Analysis With Advanced Mass Spectrometry

MS is recognized as a powerful analytical technique because it can deliver accurate and reliable identification and quantitation with molecular specificity, even for highly complex biological samples. The measurement of an analyte’s mass is incredibly informative to its identity since each element of the periodic table has a unique atomic mass and isotope distribution. So, accurately measuring the mass of an analyte ion gives extremely high confidence about how many atoms of each element it contains.

The available information about the molecule can be further expanded – and its identity confirmed – using ion fragmentation analysis, where researchers can break an ion up into various pieces and measure the masses of the fragments to confirm the bonding structure of the molecule. This process is also quantitative, meaning that the higher the abundance of a certain molecular species in the sample, the larger the signal registered by the mass spectrometer. When you consider that modern mass spectrometers can generate this type of data from just a few hundred ions, it’s easy to see that the combination of sensitivity and specificity delivered is virtually impossible to achieve with any other technique.

It’s even more impressive that MS can perform this type of analysis for biological samples that contain millions of different types of chemical species – not just for a single class of compounds. MS/MS has been applied to an incredibly broad range of biologically relevant compounds. From metabolites to intact proteins, it offers a versatile platform capable of multiplexed quantitation for 1,000s of analytes in a single run that typically takes 10s of minutes. For this reason, it has become the tool of choice for many researchers looking to understand biological phenomena at the deepest level.

Quadrupole analyzers have a much lower resolution than Time-of-Flight (ToF) or Orbitrap analyzers, so mass windows of 0.5 to 20 mass-to-charge units must be used for selection, meaning that for complex samples, chimeric MS/MS spectra are the norm rather than the exception. Matching these chimeric spectra to library reference spectra results in low-scoring matches due to the presence of unexpected spectral features. This is particularly obvious for proteomics analyses, where less than 10% of detected features are typically identifiable based on their MS/MS signature, due in large part to this chimeric spectrum challenge.

A related challenge of employing traditional MS/MS analysis with a quadrupole-ToF or quadrupole-Orbitrap, is that the quadrupole acts as a filtering device that rejects certain ions, limiting both the sensitivity and speed of analysis. Mass spectrometers can detect very low numbers of ions, but one underappreciated issue is that getting 100 ions of a certain type through the system to the detector may require starting with a million times as many molecules in the sample. This inefficiency in ion utilization is due in part to the traditional mode of operation for MS/MS, where the first MS stage operates as a filter that throws up to 99% of the ion signal away. For example, if there are 100 different target analytes of interest, then the quadrupole must be configured to select each of those mass ranges sequentially. Because ions are continuously created in the ion source, whenever one of the target ions is isolated, the quadrupole causes all ions from the other 99 targets to be filtered out of the ion beam and discarded. This means that the signal level for each target’s fragment ions is 100x lower than it could have been if the system was able to make use of all the ions available.

Practically speaking, this challenge manifests as a tradeoff that researchers must manage between the number of quadrupole isolation windows used in each analysis and the sensitivity of detection for low-level analytes within those windows. Ultimately, the number of compounds that can be detected and quantified from a complex sample is determined by how many high-quality (non-chimeric and high signal level) fragmentation spectra can be generated. 

MOBILion’s new parallel accumulation with mobility aligned fragmentation (PAMAF) technology is a novel approach to ion fragmentation analysis. It uses high-resolution ion mobility (HRIM) instead of a quadrupole filter to eliminate the tradeoff between speed and sensitivity encountered in traditional MS/MS analysis. It also serves to significantly reduce the number of chimeric spectra generated by separating isomeric and isobaric ions in the mobility dimension before fragmentation. This means that separate fragmentation spectra are created for each isomer or isobar, translating to higher quality, “pure” fragmentation spectra that are more likely to lead to accurate identifications. Researchers no longer must deal with the jumbled-up puzzle pieces, but instead, the pieces from each puzzle are pre-separated into separate boxes for easy assembly.

This enhanced performance is achieved by leveraging MOBILion’s proprietary structures for lossless ion manipulation (SLIM) technology. SLIM is a means of creating sophisticated ion processing devices using printed circuit board technology that enables miniaturization of the ion optical elements as compared to traditional MS designs. With this technology, we are also able to store up large populations of ions, so they can be saved for future analysis rather than discarded while downstream ion processing activities are occurring. This is, in essence, the “parallel accumulation” part of the PAMAF acronym.

Each packet of accumulated ions can then be pulsed into a long, serpentine ion guide path where different-sized ions take different amounts of time to travel to the MS, with typical separations occurring over a period of a few hundred milliseconds. This technique of separating ions based on how quickly they can pass through a gas under the influence of an electric field is known as ion mobility, with the resolution of the separation increasing with the path length the ions travel. MOBILion’s SLIM devices with 13 meters worth of path length can separate the full mass range of ions in a single scan, with higher resolution than any other ion mobility technology and deliver a level of specificity that is on par with the typical quadrupole isolation window – though with the added benefit of differentiating between isomers and isobars.

When coupled to a ToF mass spectrometer, this allows us to record a full precursor and fragment spectrum for every target coming from the sample without discarding any of the ion signals, describing the “mobility aligned fragmentation” part of the PAMAF acronym. This highly efficient methodology means that we can use 100% of the ion signal available, resulting in >100x better sensitivity than competitive approaches. A second benefit of using high-resolution ion mobility to deliver ions to the mass spectrometer is that the isolation happens at the speed of the ion mobility separation, meaning that the speed is no longer determined based on the speed of quadrupole analyzer electronics. Instead, we find that fragmentation spectra can be generated two-to-five times more quickly than the fastest quadrupole-based approaches. This speed boost means that PAMAF operation can enable researchers to use the same amount of time to either detect more analytes or analyze two-to-five times more samples and the high ion utilization efficiency also means that smaller sample amounts are required. This is particularly intriguing as an enabling technology for single-cell omics analysis.

The specificity of MS analysis – a measure of the identification accuracy – can be increased by incorporating additional orthogonal measurements, such as HRIM, into the workflow. High specificity gives confidence to researchers that the annotation of a certain peak is reliable because it has been characterized in numerous ways and all the measurements align with expected values for the analyte(s) of interest. PAMAF provides a way to achieve higher specificity while also increasing sensitivity, which improves the accuracy and reliability of mass spectrometric biomolecular analysis.

The anticipated impact of PAMAF on proteomics research is significant. We often hear about the fact that current MS technologies don’t have the necessary throughput to enable the population scale studies required for personalized medicine.

When the goal is to characterize millions of individuals over multiple time points, you need a tool capable of quantifying thousands of proteins – and other biomolecules – in just a few minutes. This approach provides the best opportunity to capture significant biomarkers indicative of health and overall biological status. We believe PAMAF represents the necessary step-change in MS performance whereby researchers no longer must choose speed or sensitivity but can instead achieve both simultaneously. Such an advancement could help deliver on the promises of a personalized medicine paradigm shift that would result in better clinical outcomes and health for all.

MOBILion’s new approach to MS also plays a role in drug discovery and development. Whether a researcher is working to identify a druggable target protein, elucidate a drug’s mode of action or determine the tertiary structure of a biotherapeutic, improved characterization via high-resolution ion mobility offers unique advantages over traditional MS/MS approaches that could make or break a drug program. In particular, the ability to identify mass neutral or mass similar species and create high-quality fragmentation spectra for each will significantly enhance post-translational modification (PTM) characterization workflows. In many cases, the PTMs a protein undergoes are often even more important than the sequence of the protein and detecting such nuanced changes is impossible with current technologies. We expect that applying HRIM to these problems will eliminate many current blind spots of mass spectrometry and result in pharma companies being able to develop better and safer products, faster and more efficiently. 

A significant limitation of traditional MS is its reliance on chromatographic separations, which can vary substantially across systems and change as a function of time. HRIM reduces a workflow’s reliance on chromatography, both to separate different components of a mixture and also as a means to reliably identify them based on their separation characteristics. The chemical nature of the chromatographic separations makes them subject to variations in manufacturing quality, aging of components, carryover or contamination, temperature and mobile phase composition.

MOBILion’s HRIM-based approach is a physics-based separation technique that can be calibrated and reproduced with 100x better precision than typical retention time metrics. This means that we can assign specific collision cross-section values – a measure of each ion’s size – to each analyte and then use this measurement to reliably identify the analyte over extended periods on a given system or even across different systems at various laboratories. As an analyte class agnostic technique, HRIM is well suited to extend the functionality of MS systems to achieve multiomic analyses in a way that chromatography never will, since significant hardware and method changes are required for any chromatographic system to analyze different classes of chemicals.

In fact, for low-complexity mixtures, researchers are often able to directly inject or infuse the samples and rely entirely on HRIM to simplify the resulting mass spectra, thereby eliminating the need for chromatographic separation. In cases where certain analytes may not ionize well, causing low sensitivity, researchers can employ rapid liquid chromatography (LC) methods as a means for reducing ion suppression but still rely on HRIM for peak capacity and identification. In this way, the limitations imposed by LC can be mitigated, resulting in a more robust and translatable analytical workflow where the LC fades into the background and is simply an extension of the autosampler for sample introduction.

With MOBILion’s PAMAF technology, researchers now have access to a solution that shifts the performance curve for MS analysis, delivering speed, specificity and sensitivity, while also mitigating the major pain points associated with chromatography.

We expect that the next generation of MS systems will be defined by their efficiency of ion utilization to extract every possible bit of chemical information from a sample. We are excited about working with our collaborators to explore the impact our new capabilities are having in proteomics and beyond.