Proteomics/Protein Identification - Mass Spectrometry

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[edit] Introduction

• 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.

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.(1)

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.

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• 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.

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.

[edit] References

American Society for Mass Spectrometry - What is MS?, http://www.asms.org/whatisms/p4.html

MDS Analytical Technologies - About Mass Spectrometry, http://www.mdssciex.com/products/about%20mass%20spectrometry/