Frequently Asked Questions

Analogous to genomics, which is the large-scale study of genes and genomes, proteomics is the large-scale study of all of the proteins produced by living organisms. Proteomics can encompass a large range of techniques; however, members of the PCPC primarily use mass spectrometry based techniques for the quantitation and characterization of proteins. For more information please visit our Services page.

Mass Spectrometry (MS for short) measures the mass-to-charge ratio of one or more molecules in a sample. Various MS analytical techniques can be used to determine the exact molecular weight of a molecule, provide information on the composition and structure of a compound, and can be used to quantitate compounds in simple or complex mixtures.

Multiple Reaction Monitoring (MRM, also referred to as Selected Reaction monitoring (SRM)), is a specific and sensitive mass spectrometry technique to accurately quantitate proteins of interest. Using isotopically-labeled internal standards, this technique can be used for targeted, absolute quantitation of a large numbers of proteins in complex systems within a single liquid chromatography tandem mass spectrometry (LC-MS/MS) experiment.

In a Hydrogen Deuterium Exchange (HDX) experiment, the exchangeable amide protons on the backbone of a protein are rapidly exchanged by deuterium atoms. By analyzing HDX samples on a mass spectrometer, the increased mass of the deuterium atoms can be used to accurately detect and quantitate the degree of exchange at specific sites throughout a protein. By performing these experiments over time (from 5 seconds up to several hours), it is possible to identify regions of the protein that are in defined secondary structures or have limited solvent accessibility (i.e. buried within the protein). This technique can be used to compare proteins from different batches, identify regions of a protein that become more protected when bound to a small molecule or when the protein forms a protein-protein complex, and can provide information on the secondary and tertiary structure of a protein to complement other protein structural techniques.

Analysis of proteins by mass spectrometry, such as in an HDX experiment, can be performed using two different methodologies: “bottom-up” and “top-down” proteomics. In a bottom-up experiment, proteins are digested into peptides using specific proteases (e.g. trypsin), separated by liquid chromatography, and the peptides are analyzed on a mass spectrometer. The information collected on all of the peptides can then be aligned to the known amino acid sequence, providing high-resolution (often amino acid resolution) characterization of the protein of interest. Top-down proteomics does not require prior proteoylytic cleavage of the target protein; rather, the protein is fragmented in the mass spectrometer. Although there are limitations on the size of proteins that can be measured, top-down proteomics has the benefit of being able to identify the unique proteoforms and post-translational modifications (PTMs) on individual proteins. Members of the PCPC have also perfected a hybrid approach, termed “middle-down” proteomics, which uses limited proteolysis combined with top-down proteomics, to characterize larger proteins or protein complexes that are often too large for traditional “top-down” proteomics.