Protein mass spectrometry

The application of mass spectrometry to study proteins became popularized in the 1980s after the development of MALDI and ESI. These ionization techniques have played a significant role in the characterization of proteins. (MALDI) Matrix-assisted laser desorption ionization was coined in the late 1980s by Franz Hillenkamp and Michael Karas. Hillenkamp, Karas and their fellow researchers were able to ionize the amino acid alanine by mixing it with the amino acid tryptophan and irradiated with a pulse 266 nm laser. In 1968, Malcolm Dole reported the first use of electrospray ionization with mass spectrometry. Around the same time MALDI became popularized, John Bennett Fenn was cited for the development of electrospray ionization. Koichi Tanaka received the 2002 Nobel Prize in Chemistry alongside John Fenn, and Kurt Wüthrich "for the development of methods for identification and structure analyses of biological macromolecules." These ionization methods have greatly facilitated the study of proteins by mass spectrometry. Consequently, protein mass spectrometry now plays a leading role in protein characterization. ==Methods and approaches==

Techniques 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. Protein and peptide fractionation 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 material, 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. 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 spectrometry for 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). 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. ==Applications==

Protein identification There are two main ways MS is used to identify proteins. Peptide mass fingerprinting uses the masses of proteolytic peptides as input to a search of a database of predicted masses that would arise from digestion of a list of known proteins. If a protein sequence in the reference list gives rise to a significant number of predicted masses that match the experimental values, there is some evidence that this protein was present in the original sample. Purification steps therefore limit the throughput of the peptide mass fingerprinting approach. Alternatively, peptides can be fragmented with MS/MS to more definitively identify them. MS is also the preferred method for the identification of post-translational modifications in proteins versus other approaches such as antibody-based methods. An annotated peptide spectral library can also be used as a reference for protein/peptide identification. It offers the unique strength of reduced search space and increased specificity. The limitations include spectra not included in the library will not be identified, spectra collected from different types of mass spectrometers can have quite distinct features, and reference spectra in the library may contain noise peaks, which may lead to false positive identifications. A number of different algorithmic approaches have been described to identify peptides and proteins from tandem mass spectrometry (MS/MS), peptide de novo sequencing and sequence tag-based searching. Antigen presentation Antigen presentation is the first step in educating the immune system to recognize new pathogens. To this end, antigen presenting cells expose protein fragments via MHC molecules to the immune system. Not all protein fragments bind, however, to the MHC molecules of a certain individual. Using mass spectrometry, the true spectrum of molecules presented to the immune system can be determined. Protein quantitation Multiple methods allow for the quantitation of proteins by mass spectrometry, and recent advances have enabled quantifying thousands of proteins in single cells. Protein quantification by mass spectrometry benefits from efficient sampling (counting) of many ions per protein compared to other methods. Quantifications can be performed by label-free methods and by multiplexed methods, which use isotopic mass tags as labels. Multiplexed methods can improve both quantitative accuracy and throughput. Typically, stable (e.g. non-radioactive) heavier isotopes of carbon (13C) or nitrogen (15N) are incorporated into one sample while the other one is labeled with corresponding light isotopes (e.g. 12C and 14N). The two samples are mixed before the analysis. Peptides derived from the different samples can be distinguished due to their mass difference. The ratio of their peak intensities corresponds to the relative abundance ratio of the peptides (and proteins). The first generation of methods for isotope labeling included SILAC (stable isotope labeling by amino acids in cell culture), trypsin-catalyzed 18O labeling, ICAT (isotope coded affinity tagging), and iTRAQ (isobaric tags for relative and absolute quantitation). The more recent generation of multiplexing methods include tandem mass tags (TMT) for DDA data and mTRAQ for multiplexed DIA (plexDIA). "Semi-quantitative" mass spectrometry can be performed without labeling of samples. Typically, this is done with MALDI analysis (in linear mode). The peak intensity, or the peak area, from individual molecules (typically proteins) is here correlated to the amount of protein in the sample. However, the individual signal depends on the primary structure of the protein, on the complexity of the sample, and on the settings of the instrument. Other types of "label-free" quantitative mass spectrometry, uses the spectral counts (or peptide counts) of digested proteins as a means for determining relative protein amounts. Comparing charge state distributions can give information about the structure of a protein. A wide variety of high charge states indicates disorder of the protein, whereas more compact, folded proteins result in lower charge states. By using chemical crosslinking to couple parts of the protein that are close in space, but far apart in sequence, information about the overall structure can be inferred. By following the exchange of amide protons with deuterium from the solvent, it is possible to probe the solvent accessibility of various parts of the protein. Hydrogen-deuterium exchange mass spectrometry has been used to study proteins and their conformations for over 20 years. This type of protein structural analysis can be suitable for proteins that are challenging for other structural methods. Another interesting avenue in protein structural studies is laser-induced covalent labeling. In this technique, solvent-exposed sites of the protein are modified by hydroxyl radicals. Its combination with rapid mixing has been used in protein folding studies. Proteogenomics In what is now commonly referred to as proteogenomics, peptides identified with mass spectrometry are used for improving gene annotations (for example, gene start sites) and protein annotations. Parallel analysis of the genome and the proteome facilitates discovery of post-translational modifications and proteolytic events, especially when comparing multiple species. ==References==