What is a Mass Spectrometer?
A mass spectrometer is based on the simple fact that different chemicals have different masses, and this is what determines what chemicals are present in a sample. There are many types of mass spectrometers that not only analyze the ions differently but produce different types of ions; however they all use electric and magnetic fields to change the path of ions in some way.
During the late 1980s and early 1990s, two new methods of sample ionization caused mass spectrometry to undergo a revolution in biopolymer analysis, namely ESI 38 and MALDI.39 Just over a decade ago, for the first time, mass spectrometric techniques that could measure the molecular masses of femtomole quantities of proteins in excess of 100 kDa, often to better than 0.01% accuracy, became widely available to protein chemists. These methods are both successful in forming ions from large, labile biomolecules without significant degradation of the analyte. Not only are they both sensitive techniques but also speedy – both MS and MS/MS sequence spectra can be acquired within seconds. ESI and MALDI, due to their many differences, are complementary and many biopolymer laboratories have access to both techniques.
Sample preparation: 2-Dimensional Electrophoresis and Mass Spectrometry
Sample separation, isolation and preparation for the mass spectrometric techniques generally, involves 2-DE (2-Dimenisional Electrophoresis), a technique that can separate as many as 5000 different proteins in a single experiment. In general, 2-DE is capable of separating proteins within an isoelectric point (pI) range of 3.5–10 and of molecular masses ranging from 6 to 300 kDa, as well as being able to distinguish between post-translationally modified proteins (e.g. phosphorylated) and their non-modified companions. Once separated, the protein components are revealed by staining techniques (e.g. silver stain, fluorescent stain, Coomassie blue) and once located they can then be extracted from the gel. Proteins cannot easily be eluted from gels without the use of detergents, which are detrimental to the mass spectrometric analysis; additionally, large proteins tend to be heterogeneous (e.g. as a result of glycosylation) and so possesses no single molecular mass which can be related to the corresponding entry in a database. To overcome these obstacles, the elution step tends to be accomplished after the protein has been digested by an aqueous acetonitrile wash of an excised gel piece. This increases the yield of extraction 5 and by using an in-gel digestion method employing trypsin, which has been described and is widely used as published or with minor modifications, produces a MS-compatible sample from which a protein identification can be made. The digestion step can be preceded by an optional reduction and alkylation step to cleave and block disulfide bridges that exist between cysteine residues. Robotic systems have been developed to automate the excision of protein spots from the 2D-gel, to carry out the subsequent enzymatic digestion and to transfer the samples onto MALDI-MS target plates for the initial MS analysis. For many applications, the peptides recovered from in-gel digestions require concentration and purification before being analysed by mass spectrometry especially if ESI is the ionization method used. Reversed phase high performance liquid
chromatography (HPLC) is one method of achieving this; another method involves the use of ZipTips (Millipore) (pipette tips packed with C18 material) or Poros R2 perfusion material (Perseptive Biosystems). Despite its widespread acceptance, the limitations of 2-DE include the exclusion of very small or very large proteins, very acidic or very basic proteins, as well as hydrophobic proteins such as membrane proteins. It is thought that
only 20% of the gel-loaded proteins can be visualised and analysed. Other constraints are that the amount of sample that can be loaded is limited causing the non-observance of
low concentration proteins and also that the most commonly used staining techniques have non-linear response factors. In practice, 2-DE is a labour intensive process involving manual handling steps that offer the opportunity for impurities such as keratin to contaminate the samples. One 2-D gel will take a day to complete and about one month for a complete structural analysis by MS, although automation can help both the contamination problem and speed up the analysis to some extent.
Alternative protein separation techniques
Alternative, MS-compatible methods of protein preparation are under development but no one technology has emerged as a universal replacement. These methods aim generally to interface the protein sample directly to MS/MS analyses thereby eliminating time-consuming sample preparation steps and the need for a preliminary MS analysis. Capillary isoelectric focusing (CIEF)-MS and preparative isoelectric focusing followed by size exclusion chromatography-MS are methods that have been compared in depth with 2-DE in terms of separation effciency, peak capacity, precision and robustness, with the former appearing the more favourable alternative.The direct analysis of complex peptide mixtures from a mixture of proteins using on-line, reversed phase high performance liquid chromatography (HPLC)-MS/MS has been used successfully as an alternative to 2DE and this has led onto multidimensional chromatography if greater peptide separation is desirable prior to the MS/MS analyses. Examples of two dimensional chromatography include anion or cation exchange followed by reversed phase HPLC and size exclusion chromatography followed by reversed phase HPLC. Another, alternative approach has been to use combined solid-phase micro-extraction together with capillary electrophoresis(CE) coupled to ESI MS/MS for the analysis of a total protein tryptic digest. This method afforded high resolution separation of the peptide fragments allowing the identification of 80–90% of the proteins in this particular yeast ribosome complex. Coupling microfluidic devices to MS is a further strategy that combines sample handling and separation, as well as interfacing neatly with nano-ESI-MS analysis.A different approach for selective protein fractionation and identification uses ProteinChip technology (Ciphergen) which employs a surface capture method using antibodies or chemically modified surfaces to capture specific classes of proteins which are then analysed by MALDI-MS. This technique, known as Surface Enhanced Laser Desorption Ionization (SELDI)-MS35, has great potential for the comparison of the protein content of different samples. display_block('mass_spec'); ?>