Question

Discuss the application of mass spectrometry to the analysis of proteins. Your answer should describe the most appropriate instrumentation for analysis of proteins, and discuss what kinds of information can be obtained about proteins
using this technique, including identification and partial sequencing of proteins.

Answer 1

Mass spectrometry of proteins requires that the proteins in solution or solid state be turned into an ionized form in the gas phase before they are injected and accelerated in an electric or magnetic field for analysis. The two primary methods for ionization of proteins are electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI). In electrospray, the ions are created from proteins in solution, and it allows fragile molecules to be ionized intact, sometimes preserving non-covalent interactions. In MALDI, the proteins are embedded within a matrix normally in a solid form, and ions are created by pulses of laser light. Electrospray produces more multiply-charged ions than MALDI, allowing for measurement of high mass protein and better fragmentation for identification, while MALDI is fast and less likely to be affected by contaminants, buffers and additives.^[8]

Whole-protein mass analysis is primarily conducted using either time-of-flight (TOF) MS, or Fourier transform ion cyclotron resonance (FT-ICR). These two types of instrument are preferable here because of their wide mass range, and in the case of FT-ICR, its high mass accuracy. Electrospray ionization of a protein often results in generation of multiple charged species of 800 < m/z < 2000 and the resultant spectrum can be deconvoluted to determine the protein's average mass to within 50 ppm or better using TOF or ion-trapinstruments.

Mass analysis of proteolytic peptides is a popular method of protein characterization, as cheaper instrument designs can be used for characterization. Additionally, sample preparation is easier once whole proteins have been digested into smaller peptide fragments. The most widely used instrument for peptide mass analysis are the MALDI-TOF instruments as they permit the acquisition of peptide mass fingerprints (PMFs) at high pace (1 PMF can be analyzed in approx. 10 sec). Multiple stage quadrupole-time-of-flight and the quadrupole ion trap also find use in this application.

Proteins of interest are usually part of a complex mixture of multiple proteins and molecules, which co-exist in the biological medium. This presents two significant problems. First, the two ionization techniques used for large molecules only work well when the mixture contains roughly equal amounts of constituents, while in biological samples, different proteins tend to be present in widely differing amounts. If such a mixture is ionized using electrospray or MALDI, the more abundant species have a tendency to "drown" or suppress signals from less abundant ones. Second, mass spectrum from a complex mixture is very difficult to interpret due to the overwhelming number of mixture components. This is exacerbated by the fact that enzymatic digestion of a protein gives rise to a large number of peptide products.

In light of these problems, the methods of one- and two-dimensional gel electrophoresis and high performance liquid chromatography are widely used for separation of proteins. The first method fractionates whole proteins via two-dimensional gel electrophoresis. The first-dimension of 2D gel is isoelectric focusing (IEF). In this dimension, the protein is separated by its isoelectric point (pI) and the second-dimension is SDS-polyacrylamide gel electrophoresis (SDS-PAGE). This dimension separates the protein according to its molecular weight.^[10] Once this step is completed in-gel digestion occurs. In some situations, it may be necessary to combine both of these techniques. Gel spots identified on a 2D Gel are usually attributable to one protein. If the identity of the protein is desired, usually the method of in-gel digestion is applied, where the protein spot of interest is excised, and digested proteolytically. The peptide masses resulting from the digestion can be determined by mass spectrometry using peptide mass fingerprinting. If this information does not allow unequivocal identification of the protein, its peptides can be subject to tandem mass spectrometryfor de novo sequencing. Small changes in mass and charge can be detected with 2D-PAGE. The disadvantages with this technique are its small dynamic range compared to other methods, some proteins are still difficult to separate due to their acidity, basicity, hydrophobicity, and size (too large or too small).^[11]

The second method, high performance liquid chromatography is used to fractionate peptides after enzymatic digestion. Characterization of protein mixtures using HPLC/MS is also called shotgun proteomics and MuDPIT (Multi-Dimensional Protein Identification Technology). A peptide mixture that results from digestion of a protein mixture is fractionated by one or two steps of liquid chromatography. The eluent from the chromatography stage can be either directly introduced to the mass spectrometer through electrospray ionization, or laid down on a series of small spots for later mass analysis using MALDI.