Protein Structure Analysis with Mass Spectrometry

Peptide sequencing via mass spectrometry, when performed using a bottom-up approach, is a useful and easy tool for obtaining information about primary protein structure. Such information helps elucidate the identity of that protein, or even the identities of several proteins involved in larger protein complexes.

Hydrogen deuterium exchange mass spectrometry (HDX-MS) measures the conformational changes of proteins or protein-protein interactions in protein complexes. Amide protons found within close inter- or intra-molecular contact areas often form hydrogen bonds and have different exchange rates relative to more accessible regions of the complex. By monitoring such exchanges, a protein's noncovalent structure (alone or in complex) is better understood.

Information on protein-ligand complex interactions, such as stoichiometry and dissociation constant, is often obtained using MS. Protein-ligand complexes are analyzed in the mass spectrometer in their native biological states. Mass measurement of the complexes can confirm the presence of protein-ligand interactions, while stoichiometry is determined by taking mass measurement with and without protein-ligand interactions.

Hydrogen deuterium exchange mass spectrometry (HDX-MS) aids in localization of protein-ligand interaction sites. Protein-ligand complexes are dissolved in a solution of D₂O, which enables the labile amide protons of the protein to exchange with deuterium. Typically, areas of protein-ligand interaction exchange at a much slower rate than open regions exposed to D₂O. MS is then used to determine proton exchange rates, helping to localize interaction sites and elucidate their structural information.

Crosslinking mass spectrometry (XL-MS) is used to determine protein-ligand binding sites. This process is initiated by using photoaffinity labels to photo-crosslink proteins with ligands. Samples are then enriched, digested, and analyzed by MS for ligand modifications on the protein.

Quantitative peptide‐based MS approaches can be used to determine protein stoichiometries in protein complexes, the first step in elucidating protein complex structures. Alternatively, targeted approaches with known amounts of isotopically labeled standards of the protein/peptide in question can be spiked in to gain an accurate measurement of these protein complexes.

The direct introduction of protein complexes in their native biological state enables analysis of protein-protein and protein-ligand complexes. Mass measurements coupled to protein identifications confirm the precise stoichiometry of these complexes. This information is the crucial first step in protein complex structure determination.

Crosslinking mass spectrometry (XL-MS) can be used to determine the binding stoichiometry of individual protein complexes. This is advantageous when dealing with labile proteins and protein complexes. Typically, zero length or non-specific crosslinkers are used to preserve the interacting partners for measurement of protein stoichiometry.

Native mass spectrometry (MS) plays a crucial role in generating information on protein-protein interactions, confirming protein identity, and elucidating heterogeneity data. Solvent manipulation during protein complex introduction into the mass spectrometer can reveal additional information. For example, varying solvent pH or adding organic additives can separate protein subcomplexes, which are then fragmented to confirm identity. Native MS can also give stoichiometry information on the proteins involved in protein-protein interactions.

In crosslinking mass spectrometry (XL-MS), chemical crosslinkers are used to join components of interacting complexes in order to maintain their original interaction. A bottom-up proteomics MS approach is then used. One advantage of XL-MS is that only a small sample size is required, with analysis performed directly at the proteome level. XL-MS also enables protein interactions to be examined at the physiological state of an organism, generating information that is biologically relevant.

Hydrogen deuterium exchange mass spectrometry (HDX-MS) helps localize protein-protein interaction sites. Typically, areas of protein-protein interaction have hindered exchange rates between amide protons and D₂O compared to regions with direct exposure to D₂O. Because of this difference, protein-protein interaction sites can be localized using proton exchange. MS then identifies those sites. HDX experiments are usually performed on proteins using conditions that are native to their biological state.

Liquid chromatography tandem mass spectrometry (LC-MS/MS) is the primary workflow for most researchers when performing protein identification. Proteins are enzymatically digested to their peptide components, then analyzed by LC-MS/MS. The resulting sequence data are used to determine the original protein components of the sample. For single proteins, sequence usually confirms identity. When protein complexes are involved, this top-down proteomics approach determines the individual proteins that are part of the complex.

Protein identification can be achieved without prior digestion at the intact protein level using gas phase fragmentation. This approach enables combinations of posttranslational modifications (PTMs) to be localized, permitting higher sequence coverage for individual proteins and characterization of proteoforms. Additional information, such as degradation products and sequence variants, can also be obtained within this type of experiment.

Liquid chromatography tandem mass spectrometry (LC-MS/MS) is the most frequently used approach to identify and localize PTMs on proteins and protein complexes. Multiple fragmentation methods are typically implemented during bottom-up PTM workflows to ensure complete protein characterization.

Middle-down proteomics (low-resolution studies of protein structure) involves limited proteolysis of proteins. This method permits sequence coverage of larger peptides in their native configuration, which is useful when characterizing protein-protein interactions or when probing for labile regions during protein reconstruction work for crystallization studies.

The primary advantage of top-down MS analysis is its ability to identify and localize combinations of PTMs. Using this approach, proteins are analyzed without prior digestion of their corresponding peptide species. Top-down MS analysis can involve single or multiple proteins from complex mixtures. Additional information, such as degradation products and sequence variants, is often obtained during these experiments.

Native MS is used to ascertain the expected patterns of and degree of PTM on a protein. It can also provide information on the relative abundance of modifications (e.g., glycoforms) that are present at a particular site. Due to the inherent heterogeneity and variation of attached PTMs, native MS is often performed using high-resolution, accurate-mass spectrometry.

HDX-MS can be used to study protein conformational changes induced by chemical modifications. In HDX experiments, deuterium exchange rates of unmodified and modified proteins are compared. By monitoring deuterium uptake, information is obtained on how the modification has affected the protein under study.

Specific ligands can be chemically bound to a solid support, allowing for the enrichment of certain classes of biological molecules as a function of their chemical affinity. Example molecules include phosphate, glycosyl, and ubiquitin groups. Through the use of binding ligands, specific PTMs and peptides are affinity purified for eventual MS analysis.