Protein Modifications

Understanding protein changes using mass spectrometry

The majority of proteins undergo some level of posttranslational modification (PTM) on their amino acid residues, which influences their biological function in processes such as catalysis, cell-cell signaling, and degradation. Mapping PTM sites on individual protein subunits provides information on subunit function and regulation. Within the protein complex, mapped PTMs can also help predict their partner protein interactions.

Thermo Scientific Orbitrap MS solutions enable the study and localization of PTMs. Having the flexibility of multiple fragmentation techniques also ensures complete sequence coverage and site localization of modified amino acids, as well as the modifications themselves.

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MS techniques used for protein modifications

Peptide sequencing by LC-MS/MS
Peptide sequencing by LC-MS/MS

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.

Limited proteolysis
Limited proteolysis

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.

Top-down mass spectrometry
Top-down mass spectrometry

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 mass spectrometry
Native mass spectrometry

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.

Hydrogen deuterium exchange mass spectrometry (HDX-MS)
Hydrogen deuterium exchange mass spectrometry (HDX-MS)

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.

Affinity purification mass spectrometry
Affinity purification mass spectrometry

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.

Resources

Peer-reviewed articles