shutterstock_104467115To understand protein function and mechanism of action, it is essential to determine protein complex assembly and structure. Solving the structure of large dynamic complexes often requires integrating several complementary techniques, such as mass spectrometry (MS), traditional XRD, NMR, cryo-electron microscopy (cryo-EM) and computational modeling—an approach known as integrative structural biology [1].

The primary advantage of cryo-EM is its ability to visualize the entire protein structure or protein complex assembly rather than specific regions. In recent years cryo-EM has undergone plethora of advancements contributing to the near-atomic levels of resolution achievable by this technology. MS, on the other hand, offers a means to fine-tune the data obtained with cryo-EM, providing detailed information about specific regions that might be unresolved from cryo-EM image.

Solving the structure of large dynamic complexes requires integrating several complementary MS techniques. Native MS is one such technique in a mass spectrometry toolbox. Native MS enables the study of intact protein, non-covalent protein-protein and protein-ligand complexes in their biological state. For protein complexes, high resolution accurate mass (HRAM) measurements are taken confirm the presence of the complexes and additional information, such as stoichiometry of the individual subunits and dissociation constants, can be determined. For single protein, native MS can also be used to examine expected pattern and degree of posttranslational modifications (PTMs) purity and heterogeneity. It can provide relative abundance of these modifications such as the various glycoforms that are present at a particular site.

 What is the Q Exactive UHMR mass spectrometer?

The Q Exactive UHMR MS is Thermo Scientific’s flagship mass spectrometer (MS) for performing Native MS and is specifically designed with the needs of structural biologists in mind. The instrument combines increased sensitivity and mass resolution at high m/z (up to m/z 80,000), with MS2 and pseudo-MS3 capabilities – creating a unique and powerful analytical solution for Native MS and top-down Native MS. The instrument’s unique design and capability enables the investigation of structure and heterogeneity of mega Dalton biomolecules and macromolecular protein complexes, similar to the mass range of analytes submitted to structural analysis by cryo-EM. It provides unprecedented structural detail which you cannot see with other methods enabling deeper understanding of protein function, disease mechanisms, potential drug targets, and bio-therapeutic compounds.

What are the features and benefits of the Q Exactive UHMR mass spectrometer?

The Q Exactive UHMR mass spectrometer, provides high resolution (up to 200,000 (FWHM) at m/z 400) and orders of magnitude enhanced sensitivity at high m/z compared to other HRAM mass spectrometers. This allows the instrument to analyze intact mega-Dalton protein assemblies and resolve small differences in masses that reveal key ligands, modifications and interactions. The Q Exactive UHMR MS taps into two main hardware modifications, one of them is in-source trapping capability that enables improved transmission and controllable desolvation and fragmentation right after the RF-lens where the molecules enter the mass spectrometer. With this feature users gain detailed structural insights for deeper understanding of biological processes as it enables pseudo MS3 or direct ability to perform top down of individual subunits. The other is high mass quadrupole selection (up to m/z 25,000) and higher HCD fragmentation efficiency for native top-down analysis. This can enable identification of the protein or protein complex, provide insights on modifications.

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How can the Q Exactive UHMR mass spectrometer assist Cryo-EM users?

In most cryo-EM experiments prior knowledge about the sample is required before analysis. Information such as purity, heterogeneity, integrity and identity of the sample is needed in order to obtain meaningful structural information. For example, highly pure samples are needed to generate cryo-EM images. This is due to the fact that cryo-EM images contain low signal-to-noise ratio, resulting in images that are very noisy with little contrast. Data analysis requires picking of the particles, associating particles into structurally identical groups, and then averaging grouped particles into a high resolution three dimensional structure. When the proteins are pure, fewer mistakes are made during the grouping process. Additionally, heterogeneity within the sample such as presence of additional molecules other than the protein or protein complex of interest can also hamper cryo-EM analysis by suppressing the ability to obtain high resolution structures.

While crosslinking MS is a common practice to validate cryo-EM structures, native MS is rapidly gaining ground as a screening technique for cryo-EM samples. The Q Exactive UHMR is designed with this in mind, allowing high speed (experiments take seconds to minutes) screening of small protein samples all the way up to intact virus particles. The current standard for cryo-EM sample screening is negative stain that gives the user an idea of the global shape of the complex and how uniform the particles are. Native mass spectrometry on the other hand will not only provide the user with an idea of the heterogeneity of the sample, but also if there are any post translational modifications present (which are often not visible in cryo-EM), small molecule or ligand binding, the presence of endogenous lipids and the topology of the protein complex all in a single experiment that only takes minutes. Thus allowing for better informed decisions on which samples are actually worthwhile spending valuable cryo-EM time on. On top of this, native mass spectrometry requires the same sample conditions (1-4 ul of sample at a concentration of 1 mg/ml) as cryo-EM so no additional preparations have to be made). Currently, countless hours and days are spent obtaining this information. Native mass spectrometry is a faster and cheaper solution to the issue of EM sample screening. Combined with the fact that native mass spectrometry is able to provide orthogonal structural information, native MS and cryo-EM are a match made in heaven!

What are the benefits from using the Q Exactive UHMR mass spectrometer?

The combined results from mass spectrometry and cryo-EM result in a more accurate, more complete and more reliable atomic model of biomolecular complexes.

  • Q Exactive UHMR MS is a unique solution to the pain points encountered by research and industrial customers in structural biology and biopharma research.
  • It’s designed for those scientists who need higher resolution and sensitivity in the ultra-high mass range and the ability to perform highest quality native MS and native top-down experiments.
  • Its unique combination of high resolution, high sensitivity and MS2/pseudo-MS³ capabilities makes it ideal for these applications.
  • It’s the perfect solution for native top-down characterization of protein complexes, enabling new insights into native protein structure and protein interactions.
  • It offers the unparalleled ability to resolve and characterize co-occurring assemblies, to resolve small differences in masses that reveal key ligands, modifications and interactions.

 

[1] https://www.fei.com/integrative-structural-biology/