Proteomics/Protein Identification - Mass Spectrometry< Proteomics
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- Mass Spectrometry Overview
Mass spectrometry is a technique in which gas phase molecules are ionized and their mass-to-charge ratio is measured by observing acceleration differences of ions when an electric field is applied. Lighter ions will accelerate faster and be detected first. If the mass is measured with precision then the composition of the molecule can be identified. In the case of proteins, the sequence can be identified. Most samples submitted to mass Spectrometry are a mixture of compounds. A spectrum is acquired to give the mass-to-charge ratio of all compounds in the sample. Mass spectrometry is also known as 'mass spec' or MS for short. Mass spectrometry throws light on molecular mechanisms within cellular systems. It is used for identifying proteins, functional interactions, and it further allows for determination of subunits. Other molecules in cells such as lipid components can also be defined.
A mass spectrometer is composed of several different parts: a source that ionizes the sample, the analyzer that separates the ions based on mass-to-charge ratio, a detector that "sees" the ions, and a data system to process and analyze the results. You can also measure relative abundance of an ion using mass spectrometry. Different compounds have differential ionization capabilities and therefore intensity of your ion is not a direct correlation to concentration.
Mass spectrometry can be a high throughput analytical method due to the ability for a mass spectrum to be measured rapidly and with minimal sample handling as compared to gel methods.
It is an analytical method which has a variety of uses outside of proteomics, such as isotope and dating, trace gas analysis, atomic location mapping, pollutant detection, and space exploration
- History of Mass Spectrometry
The history of this technique finds its roots in the first studies of gas excitation in a charged environment, more than 100 years ago. This pioneering work led to the identification of two isotopes of neon (neon-20 and neon-22) via mass to charge ration discrimination by J.J Thomson in 1913. Over the next fifty years the fundamental basis of the technique was further developed. After the coupling of gas chromatography to Mass Spectroscopy in 1959 by researches at Dow Chemical, the full potential of the technique as a highly accurate, quantitative method for exploring compounds was realized, spurring a wave of developments which continue to the present day. The precision of mass spectrometry led to the discovery of isotopes.
- Implications of Mass Spectrometry for Proteomics Applications
The technique of mass spectrometry is a valuable tool in the field of proteomics. It can be used to identify proteins through variations of mass spectrometry techniques. The most common first approach to proteomics is a bottom-up approach in which the protein is digested by a protease, such a trypsin, and the peptides are then analyzed by peptide mass fingerprinting, collision-induced dissociation, tandem MS, and electron capture dissociation. Once the peptides masses have been determined the mass list can be sent to a database, such as MASCOT, where the list is compared to the masses of all known peptides. If enough peptides match that of a known protein you can identify your protein. If the masses of your peptides do not match a known protein you can sequence your peptide by de novo sequencing using MS/MS methods; where you isolate your peptide and break it along the peptide bond backbone forming y and b ions from which you can determine the sequence. The advantages of the bottom-up approach are that the small size of tryptic peptide ions is easy to handle biochemically than entire protein ions because of their relatively small masses that are easier to be determine. Beside bottom-up approach, another approach is top-down. In top-down approach, the complete proteins are directly analyzed by using mass spectrometer without solution digestion as bottom-up does. The advantages of the top-down approach are that it can sometime provide the complete coverage of the protein. But since whole proteins are hard to handle biochemically compared to small peptide pieces, it makes top-down approach difficult to analyze.
Another use of mass spectrometry in proteomics is protein quantification. By labeling proteins with stable heavier isotopes you can in turn determine the relative abundance of proteins. Companies now produce kits, such as iTRAQ (Applied Biosystems), in order to do this at a high-throughput level.
One of the most powerful ways to identify a biological molecule is to determine its molecular mass together with the masses of its component building blocks after fragmentation. There are two dominant methods for doing this. The first is electrospray ionization (ESI), in which the ions of interest are formed from solution by applying a high electric field. This is done by applying a high electric field to the tip of a capillary, from which the solution will pass through. The sample will be sprayed into the electric field along with a flow of nitrogen to promote desolvation. Droplets will form and will evaporate in a vacuumed area. This causes an increase in charge on the droplets and the ions are now said to be multiply charged. These multiply charged ions can now enter the analyzer. ESI is a method of choice because of the following properties: (1)The "softness" of the phase conversion process allows very fragile molecules to be ionized intact and even in some non-covalent interactions to be preserved for MS analysis. (2)The eluting fractions through liquid chromatography can then be sprayed into the mass spectrometer, allowing for the further analysis of mixtures. (3)The production of multiply charged ions allow for the measurement of high-mass biopolymers. Multiple charges on the molecule will reduce its mass to charge ratio when compared to a single charged molecule. Multiple charges on a molecule also allows for improved fragmentation which in turn allows for a better determination of structure. The second is matrix-assisted laser desorption/ionization (MALDI) in which the molecular ions of interest are formed by pulses of laser light impacting on the sample isolated within an excess of matrix molecules. This enables the determination of masses of large biomolecules and synthetic polymers greater than 200,000 Daltons without degradation of the molecule of interest. The advantages of MALDI are its robustness, high speed, and relative immunity to contaminants and biochemical buffers.
A type of mass spectrometer often used with MALD is TOF or Time of Flight mass spectrometry. This enables fast and accurate molar mass determination along with sequencing repeated units and recognizing polymer additives and impurities. This technique is based on an ultraviolet absorbing matrix where the matrix and polymer are mixed together along with excess matrix and a solvent to prevent aggregation of the polymer. This mixture is then placed on the tip of a probe; then the solvent is removed while under vacuum conditions. This creates co-crystallized polymer molecules that are dispersed homogeneously within the matrix. A pulsing laser beam is set to an appropriate frequency and energy is shot to the matrix, which becomes partially vaporized. In turn the homogeneously dispersed polymer within the matrix is carried into the vapor phase and becomes charged. To obtain a superb signal-to-noise ratio, multiple laser shots are executed. The shapes of the peaks are improved and the molar masses determined are more accurate. Fianlly,in the TOF analyzer the molecules from a sample are imparted identical translational kinetic energies because of the electrical potential energy difference. These ionic molecules travel down an evacuated tube with no electrical field and of the same distance. The smallest ions arrive first at the detector, which produces a signal for each ion. The cumulative data from multiple laser shots yield a TOF mass spectrum, which translates the detector signal into a function of time, which in turn can be used to calculate the mass of the ion.
In addition to these ionization techniques, highly powerful mass analyzers have been developed. These analyzers measure the mass/charge ratio of intact ionized biomolecules, as well as their fragmentation spectra, with high accuracy and high speed. The measurement of fragmentation spectra is called tandem MS or MS/MS. In conjunction with single stage MS (with intact precursor ions) tandem MS can be utilized to help elucidate a protein since the problem of elucidation will reduce to assembling the puzzle pieces of the fragmented protein.
American Society for Mass Spectrometry - What is MS?, http://www.asms.org/whatisms/p4.html
Mass Spectrometry in the Postgenomic Era Annual Review of Biochemistry Vol. 80: 239-246 (Volume publication date July 2011) DOI: 10.1146/annurev-biochem-110810-095744 https://ted.ucsd.edu/webapps/portal/frameset.jsp?tab_tab_group_id=_2_1&url=%2Fwebapps%2Fblackboard%2Fexecute%2Flauncher%3Ftype%3DCourse%26id%3D_767_1%26url%3D
University of Illinois at Urbana-Champaign School of Chemical Sciences http://scs.illinois.edu/massSpec/ion/esi.php
University of Southern Mississippi School of Polymers and High Performance Materials http://www.psrc.usm.edu/mauritz/maldi.html