Van Eyk, Dunn - Proteomic and Genomic Analysis of Cardiovascular Disease - 2003 (522919), страница 56
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EI and CI are most suitable for small volatile substances, however, the latter two processes were widely used in bioanalytical laboratories duringthe 1980’s and early 90’s [3, 4]. This was the era preceding the advent of the nowperhaps more familiar processes of MALDI and ESI. These two techniques nowdominate scientific literature concerned with the analysis of large biomolecules.Karas and Hillencamp are credited with the development of MALDI [5]. Here,analyte solutions are allowed to dry onto stainless steel target plates in the presence of a matrix material.
This causes the formation of co-crystals. The matrix isgenerally a low molecular weight organic acid chosen for it’s characteristic absorption spectra. Hence, by firing laser light at the target, a gaseous plume of ions iscreated because, in simple terms, the energy of the beam is absorbed by the matrix and transferred to the analyte molecules, which become charged and cantherefore be accelerated into the analyser.Proteomic and Genomic Analysis of Cardiovascular Disease.Edited by Jennifer E.
van Eyk, Michael J. DunnCopyright © 2003 WILEY-VCH Verlag GmbH & Co. KGaA, WeinheimISBN: 3-527-30596-319612 Mass SpectrometryESI is an atmospheric ionisation technique, which can be used to analyse awide range of polar molecules. Pioneered by Fenn et al. [6] in 1985, its first application to protein analysis was published four years later [7].
The mechanism ofESI involves the emission of ions from a droplet into the gas phase at atmospheric pressure. Typically the analyte-containing solution is passed directly into astainless steel capillary, which carries a high potential, normally set at 3 to 5kV.The strong electric field and action of nebulising nitrogen gas causes the formation of a fine spray containing highly charged droplets at the tip of the capillary.These charged droplets are desolvated in a heated source causing the emission ofions.
The ions are then transferred from atmospheric pressure into the mass analyser. ESI is a “soft” ionisation technique and typically gives rise to mass spectradominated by protonated molecules for positive ion analysis and deprotonatedmolecules for negative ion analysis. Higher molecular weight compounds such asproteins can produce a series of multiply charged ions, which can be mathematically deconvoluted to determine a pseudo-molecular ion species [8]. The presenceof contamination, such as ammonium or sodium salts, may cause some compounds to form adducts.
In addition to the [M+H]+ molecular ion, resulting spectra show adduct ions such as [M+NH4]+, [M+Na]+ and [M+K]+. These adducts areobserved 17, 22 and 38 Da, respectively up from the protonated molecular ion.Typical flow rates for ESI are in the range 5 ll/min to 200 ll/min althoughsources can be operated easily up to 1 ml/min. However, when attempting to analyse low abundant proteins and peptides there are key advantages of using muchlower flowrates. Results are dependent on the concentration of the sample in thesolvent rather than the total amount of sample entering the source.
Wilm andMann built a miniaturised electrospray device allowing analytes to be introducedinto the instrument at nanolitre flowrates [9]. This development meant that peptide samples could be analysed more efficiently because the droplets produced are*100 times smaller in volume than those in conventional electrospray sources.The resulting yield of ions is far more efficient therefore reducing the totalamount of protein required. Initially fitted onto a triple quadrupole instrument,the nanospray device soon became popular.
Typically the peptide mixtures weredesalted and pre-concentrated using a small glass capillary packed with Poros R2resins [10–12]. Additionally, increased sensitivity can be achieved by coupling ESImass spectrometry with liquid chromatography (LC) and this will be discussed indetail later in the chapter.12.2Mass AnalysersOnce the ions have been created they enter the analyser where they are separatedand mass analysed. Time of flight tubes, quadrupoles and iontraps are all types ofanalysers and these can be arranged in a number of configurations to allow complicated scan functions to be performed. For example MS/MS experiments wheretwo steps of mass spectrometry are performed in tandem. This generally involves12.2 Mass Analysersselection of a particular precursor ion, which enters a collision cell to be fragmented by colliding under low pressure with argon or nitrogen.
This process is calledCollision Induced Dissociation (CID). Resulting fragment ions are subsequentlymeasured using a second mass analyser.Perhaps the simplest way to separate ions is via their time of flight. TOF analysers are commonly used in conjunction with MALDI and rely on digitisation frequencies of 20 Hz or greater to allow very accurate timing of the ions. Smallerions travel faster and arrive at the detector before the heavier ions.
The KineticEnergy (KE) of each ion is proportional to the square route of it’s mass (M) as given by the formula KE = ½ MV2, where ion velocity is V. Simple calibration routines using known compounds are used to convert time to m/z to give a massspectral representation. Reflectron devices are simple electronic mirrors which improve resolution by increasing the flight time of the ions.A quadrupole analyser consists of a set of four steel rods. Opposing rods have aradio-frequency (rf) and a dc voltage applied to them. The two sets of rods are1808 out of phase.
All ions entering the analyser take a helical path through therods with the dc function only allowing ions of a particular mass to pass throughand be detected. A triple quadrupole analyser is essentially three sets of quadrupole rods arranged one after another. This arrangement allows Tandem MS/MSexperiments to be performed.The mid 90’s saw the development of hybrid instruments such as the Q-Tof(Micromass) and QStar (Applied Biosystems). These instruments offer advantagesin terms of sensitivity and resolution.
The use of a reflectron time of flight analyser replaces the third quadrupole enabling more ions to be sampled in a shortertimeframe (duty cycle) thus greater sensitivity is achieved. The reflectron devicealso separates ions more effectively than a quadrupole and thus it is possible toobtain resolution of 10,000 or better [13]. MS/MS spectra of peptides can thereforebe acquired with each fragment ion resolved into individual isotopes. This is extremely important if peptide sequencing is to be undertaken accurately. The massdifference between amides and carboxylic acid groups is easily recognised so thatasparagines and aspartic acids, glutamines and glutamic acids can be readily assigned.
It is even possible to differentiate glutamine and lysine residues, whichdiffer in mass by 0.03638 Da [14]. Isobaric residues leucine and isoleucine are notnormally distinguished although this can be achieved by high energy CID using aTof-Tof analyser [15].Iontrap mass analysers are similar to quadrupoles and consist of a ring electrode and two endcaps. Ions, produced in the ion source, are effectively stored inthe trap. In MS mode these ions are sequentially ejected and scanned onto the detector to produce a full mass spectrum.
In MS/MS mode the ions produced bythe source are again stored in the ion-trap. At this stage, all ions, except the preselected precursor ion, are ejected from the trap. The remaining precursor ionsare then excited, causing them to collide with background helium atoms, and toundergo fragmentation. The resultant fragment ions are then sequentially ejectedand scanned onto the detector to produce an MS/MS spectrum. Further fragmentation of the fragment ions, to give MS3 (MS/MS/MS) spectrum, is also possible.19719812 Mass Spectrometry12.3 Strategies for Protein and CharacterisationThere are two key factors, which drive the development of new instruments. Improvements in overall sensitivity and resolution are always welcome, although thistypically impacts on the overall cost of the machinery.
Many proteomics laboratories are geared towards high throughput analyses and so the speed at which measurements can be made is also important.12.3Strategies for Protein and CharacterisationMass spectrometry may be applied to answer many questions and there are several ways to address protein characterisation using mass spectrometry (Fig. 12.1).The choice of technology primarily depends on the complexity of the problem andthe amount of protein available. The emergence of proteomics has seen a vast increase in the number of proteins requiring analysis. In 1993 several groups reported on the use of mass spectrometry-derived data to interrogate protein sequence databases [16–19].
Since this time methodologies for the identification oflow levels of proteins separated by 1-D or 2-D gel electrophoresis have improved[20]. Nowadays it is routinely possible to analyse as little as 5 ng of starting material (equating to 100 femtomoles for a 50 kDa protein), from CBB or silver-stainedgels. Typically, proteins of interest are excised from the gel, washed, reduced andalkylated and enzymatically digested using trypsin.
An advantage of this methodis that detergents, salts and reducing agents are eliminated during the process. Itis important to minimise contamination from keratin as this gives rise to peptideswhich can dominate the resulting mass spectrum. Other strategies involve digesting the proteins in solution or on blots [21, 22]. Following digestion a variety ofanalytical routes are available as indicated in the schematic. The following sectionwill describe each technique in more detail illustrated with examples.12.3.1Protein Identification by MALDI-TOFMALDI-TOF mass spectrometry can be used as a “first pass” to generate PeptideMass Fingerprints (PMF’s) for enzymatically digested protein samples.
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