Protein mass spectrometry

Protein mass spectrometry refers to the application of mass spectrometry to the study of proteins. Mass spectrometry is an important method for the accurate mass determination and characterization of proteins, and a variety of methods and instrumentations have been developed for its many uses. Its applications include the identification of proteins and their post-translational modifications, the elucidation of protein complexes, their subunits and functional interactions, as well as the global measurement of proteins in proteomics. It can also be used to localize proteins to the various organelles, and determine the interactions between different proteins as well as with membrane lipids. Protein mass spectrometry refers to the application of mass spectrometry to the study of proteins. Mass spectrometry is an important method for the accurate mass determination and characterization of proteins, and a variety of methods and instrumentations have been developed for its many uses. Its applications include the identification of proteins and their post-translational modifications, the elucidation of protein complexes, their subunits and functional interactions, as well as the global measurement of proteins in proteomics. It can also be used to localize proteins to the various organelles, and determine the interactions between different proteins as well as with membrane lipids. The two primary methods used for the ionization of protein in mass spectrometry are electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI). These ionization techniques are used in conjunction with mass analyzers such as tandem mass spectrometry. In general, the protein are analyzed either in a 'top-down' approach in which proteins are analyzed intact, or a 'bottom-up' approach in which protein are first digested into fragments. An intermediate 'middle-down' approach in which larger peptide fragments are analyzed may also sometimes be used. 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 80's 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. Though important, the breakthrough did not come until 1987. In 1987, Koichi Tanaka used the 'ultra fine metal plus liquid matrix method' and ionized biomolecules the size of 34,472 Da protein carboxypeptidase-A. 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. 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. 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-trap instruments. 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. Tandem mass spectrometry (MS/MS) is used to measure fragmentation spectra and identify proteins at high speed and accuracy. Collision-induced dissociation is used in mainstream applications to generate a set of fragments from a specific peptide ion. The fragmentation process primarily gives rise to cleavage products that break along peptide bonds. Because of this simplicity in fragmentation, it is possible to use the observed fragment masses to match with a database of predicted masses for one of many given peptide sequences. Tandem MS of whole protein ions has been investigated recently using electron capture dissociation and has demonstrated extensive sequence information in principle but is not in common practice. In keeping with the performance and mass range of available mass spectrometers, two approaches are used for characterizing proteins. In the first, intact proteins are ionized by either of the two techniques described above, and then introduced to a mass analyzer. This approach is referred to as 'top-down' strategy of protein analysis as it involves starting with the whole mass and then pulling it apart. The top-down approach however is mostly limited to low-throughput single-protein studies due to issues involved in handling whole proteins, their heterogeneity and the complexity of their analyses.