Mass spectrometry can be used to determine both primary and higher order structures of proteins. The basis for these investigations lies in the ability of mass analysis techniques to detect changes in protein conformation under differing conditions. These experiments include:

• Monitoring charge states of proteins
• Monitoring charge states in combination with proteolytic digestion
• Monitoring charges states with chemical modification

The accuracy and sensitivity of PMF allows for the exploration of protein structure and even structural dynamics.

PMF combines enzymatic digestion, mass spectrometry, and sequence specific data analysis to produce and examine proteolytic fragments. This information could then be used to identify protein and get information about protein structure.

Higher order structure of a protein can be evaluated when PMF techniques are combined with limited proteolytic digestion. Limited proteolysis refers to the exposure of a protein or complex to digestion conditions that last for a brief period. This is performed to gain information on the parts of the protein exposed to the surface.

The sequence specificity of the proteolytic enzyme plays a major role in the application of mass spectrometry to protein structure. A sequence specific protease reduces the number of fragments that are produced and, concomitantly, improves the likelihood for statistically matches. Accessibility and flexibility of a protein is also very important. Surface proteins are usually hydrophilic so proteases that cleave hydrophilic sites are preferred.

Recognizing conformational changes

PMF can be used to recognize simple conformational differences between protein states. After conformational changes, the same protein would digest into different mass maps.

ESI has been used to monitor protein folding and protein complexes. Some proteins exhibit a distinct difference in their charge state distribution which is a reflection of their solution conformation. ESI is a simple but highly sensitive and informative method to characterize the functional shape(s) of proteins (globular or extended) prior to more material-intensive and time-consuming spectroscopic or crystallographic studies.