Mass Spectrometry

Recent advances in mass spectrometric techniques for moderate to large biomolecules allow high-resolution mass information to be obtained for many of the (metallo)enzymes of interest. The Department of Chemistry and the MRIL have an excellent mass spectrometry facility including electrospray ionization quadrupole and matrix-assisted laser desorption time-of-flight instruments [I. Jonathan Amster, UGa].

Acronyms, synonyms

• Mass Spectrometry
• Matrix Assisted Laser Desorption/Ionization MS
• Electrospray Ionization MS
• Fast Atom Bombardment MS

Measured physical quantities

• Mass-to-charge ratio of singly charged or multiply charged molecular ions and fragment ions

Information available

• Molecular weight
• Primary structure of peptides and proteins
• Some aspects of quaternary structure can be derived

Information NOT available, limitations

• Peptide, protein secondary and tertiary structure cannot be directly determined, although some inferences can be made

Examples of questions that can be answered

• What are the sites of post-translational modification in this protein? What is the nature of the modification?
• Has a mutant protein been prepared properly? Is the desired change in amino acid sequence occurring at the expected position?
• What are the molecular weights and sequences of peptides in an enzymatic digest?

Major advantages

• Provides molecular weights of peptides and proteins with high accuracy (0.1-0.01%)
• Highly sensitive; requires fmol-pmol quantities of sample
• Sample purity not important
• Can be coupled with on-line separation methods such as HPLC and capillary electrophoresis for the analysis of mixtures

Major disadvantages

• Noncovalent complexes are often disrupted
• Cannot distinguish stereoisomers
• Expensive instrumentation

Sample constraints

• Few for proteins and peptides, as purity and sample requirements are not restrictive (fmol-pmol quantities can be used)
• Other biopolymers (nucleic acid, carbohydrates) are more difficult to analyze