Van Eyk, Dunn - Proteomic and Genomic Analysis of Cardiovascular Disease - 2003 (522919), страница 57
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This peptide mass mapping approach was first reported by Henzel and co-workers [16].Each PMF represents a collection of accurately determined peptide molecularweights, which can be submitted as a database search query (Fig. 12.2). To improve sensitivity, the sample supernatants can be desalted and concentrated usingcommercially available ZipTips [23] prior to target loading.
These miniature columns are packed with a small amount of C18 reversed phase resin.3Fig. 12.1 Schematic to show a general overview of strategies for protein identificationand characterisation using mass spectrometry.19920012 Mass Spectrometry12.3 Strategies for Protein and CharacterisationNot all proteins can be identified directly by PMF’s. For example, if the protein sequence is not present in the database or if the spectrum only contains a limitednumber of peptides. In the latter cases, PSD may be used to confirm ambiguousprotein identifications. Here, a particular peptide molecular ion may be selectedusing an iongate and the fragments produced as a consequence of the high energy ionisation process, can be mass analysed by focusing them at the detector(Fig. 12.2).
Typically PSD spectra contain complex patterns of fragment ions,which are difficult to interpret. Although such data may yield the identity of a putative protein hit, PSD is not generally used to sequence peptides. Additionally,MALDI-TOF is less effective for the analysis of complex protein mixtures as generally only the most abundant proteins are identified. Small proteins (20 kDa orless) may also prove difficult to analyse as these tend to generate fewer appropriately sized tryptic peptides therefore decreasing the chances of an unambiguousidentification. In such cases further analytical techniques must be employed.12.3.2Protein Identification by Tandem MS/MSTandem MS/MS of peptides may be used to provide sequence information by virtue of the fragment ions produced (Fig.
12.3). Fragmentation occurs generallyacross the peptide bond leading to a ladder of sequence ions that are diagnostic ofthe amino acid sequence. The difference between consecutive ions in a series indicates the mass of the amino acid at that position in the peptide. The most common ion types are b and y ions. The C-terminal containing fragments are designated y-ions and the N-terminal containing fragments are designated b-ions [24].Peptides created by trypsin proteolysis and ionised by electrospray generally formions that are doubly charged.
This stems from the presence of basic groups within the peptide, namely, the alpha amino group at the N-terminus and the sidechain of the C-terminal lysine or arginine. MS/MS spectra of such peptides generally yield a prominent y-type ion series in the high mass end of the spectrum [25].Ideally, for de novo sequencing purposes, a complete set of complementary b andy ions will ensure a high confidence level for the proposed peptide sequence.Peptides derived from gel separated proteins can be delivered into the massspectrometer either by using a static nanoelectrospray device or, more dynamical3Fig. 12.2 A – MALDI-TOF PMF of a 21 kDaspot from human heart left ventricle.
Thespectrum shows isotopically resolved molecular ions for the peptides resulting from tryptically digested material. Following internal lockmass calibration, the spectrum can be processed to give a monoisotopic masslist,which is then submitted as a database searchquery. B – The results of the database searchdisplayed via a web browser.
In this instancethe protein is clearly identified as myosinlight chain 1 with 10 peptides matched withina mass tolerance of 30 ppm. The peptidescover 62% of the total sequence. C – PSDspectrum of m/z 1501.85 showing the fragment ions matching the sequenceDTGTYEDFVEGLR. PSD data can be used toverify an ambiguous protein identity.
Datacourtesy of Nicola Leeds.20120212 Mass SpectrometryFig. 12.3 A – MS/MS spectrum of a peptideoriginating from phosphoglycerate mutase(P15 259; PGM), PGM was isolated from a 1D gel band reduced, alkylated and digestedwith trypsin. Peptides were separated using a75 micron ID PepMap RP column and elutedat a flowrate of 200 nl/min into an ESI sourcefitted to a Q-Tof instrument. The doublycharged peptide ion at m/z 679.26, relating toMr 1356.58 Da, was selected for MS/MS. B –The fragment ions produced matched the sequence HGESTWNQENR. A strong series ofb and y ions are matched to this sequence.The presence of the histidine at the N-terminus helps to stabilise the b ions.ly, by liquid chromatography.
The on-line coupling of LC to Tandem MS/MS instrumentation (LC/MS/MS) is presently unparalleled in terms of sensitivity anddynamic range and is particularly powerful when used in conjunction with massspectrometers capable of data dependent scanning. Automatic switching, for example, from survey mode to MS/MS mode, enables a great deal of data to be generated in a short space of time.LC/MS/MS datasets can contain hundreds of MS/MS spectra and these are typically processed into a format to allow direct database query submissions.LC/MS/MS can be used alone or in combination with 1-D or 2-D electrophoresis.
RP chromatography is the method of choice due to the solvents being compatible with the ionisation process. Sample cleanup, separation and concentration12.3 Strategies for Protein and CharacterisationFig. 12.4 Schematic showing the valve configurations for an LC system set up to pre-concentrate the peptide sample. This system canbe linked to an autosampler capable of injecting ll amounts of sample. Valve positions areshown for A) sample injection B) sampleloading onto the pre-column and C) switchingthe analytical column in line with the pre-column.20320412 Mass Spectrometryare all achieved in a single experiment with the temporal separation making itpossible for complex mixtures of peptides to be analysed effectively.
Nowadays,50–100 lm ID columns, packed with polymeric or silica based C18 coated stationary phase, are preferred as these minimise the elution volume of peptides andhence increase the concentration of the peptides entering the mass spectrometer.To improve sensitivity, pre-concentration techniques can be employed. For example, the sample can be concentrated on a pre-column of RP media, prior toseparation on the analytical column. This is made possible due to an elegant valveswitching system (Fig.
12.4). The pre-column not only concentrates the samplebut also removes salts, buffers and other low molecular contaminants. This helpsto preserve the life time of the analytical column. Other advantages of such a system include the ability to automate sample introduction, via autosamplers capableof injecting microlitre volumes.To enhance the MS/MS information obtained for low level peptides the abilityto selectively extend the analysis time of interesting peaks has been demonstrated[26]. When a peak of interest is eluted the flow rate is reduced so as the timespent acquiring data on the peptides is increased. This is referred to as “peakparking” and flow rates between 5–200 nl/min are typically employed. CZE canalso be coupled to Tandem MS/MS instrumentation [27], although this techniqueis less robust than LC/MS/MS and consequently less popular.Multidimensional LC is beginning to emerge as an alternative approach to 2-DE[28, 29].
Generally, this combines strong cation exchange (SCX) chromatographyand RP chromatography. Discrete fractions of peptides are displaced from theSCX column onto the RP column using a salt gradient. Peptides are retained onthe RP column and contaminating salts and buffers are washed to waste. Thepeptides are then eluted from the RP column into the mass spectrometer. Coupling direct-identification approaches with quantitative methods, such as ICAT[30] and MCAT [31], to measure relative protein expression will greatly increasethe value of the data produced. Even when analysing complex mixtures usingmultidimensional LC, pre-fractionation is still beneficial because, as with any direct analysis of crude protein mixtures, the major proteins present are likely tomask the less abundant proteins.
Multidimensional LC also has the ability to detect and identify a wide variety of protein classes, including proteins with extremepI’s, Mr abundance and hydrophobicity (e.g. Transmembrane proteins).12.3.3Database SearchingThe recent advances in mass spectrometry technologies have been closely followed by the development of bioinformatics tools to extract the relevant information contained within information-rich datasets. Programmes such as MASCOT,Protein Prospector, Sequest and Global server [32–35] are invaluable and by accepting uninterpreted data, make the task of analysing large datasets much moreamenable. De novo sequencing algorithms hold promise however, in our laboratory, this task still remains largely a manual process. Typically, MS/MS data is ini-12.3 Strategies for Protein and Characterisationtially searched against protein sequence databases and subsequently against nucleotide databases, such as expressed sequence tag (EST), and more recently rawgenomic sequences [36, 37].
There are several advantages of searching genomicdatabases directly. The MS/MS data may help to define the structure of the geneby locating start and stop codons and also intron-exon boundaries. Unfortunately,nucleotide databases cannot be interrogated effectively by MALDI alone. Modifications of proteins, that are not apparent from the DNA sequence, can also be identified using proteomic methodologies.12.3.4Post Translational ModificationsOnce translated, protein molecules are transported through the cell and many become modified to suit particular tasks or functions. Such modifications includedisulphide bridge formation, the addition of lipids, glycosylation and phosphorylation. It is these modification events which greatly diversify the proteome.Although there are only 30 000 gene products encoded within the human genome[38] each gene could potentially produce a heterogeneous population of relatedprotein molecules, perhaps hundreds of unique species.
Whilst minimal sequenceinformation is sufficient to identify a protein in a sequence database, perhaps bymatching two or three peptides, the analysis of post translational events can befar more challenging. Ideally, the goal is to obtain complete sequence coverage ofthe protein and hence more intricate sample preparation and analyses are required.It is possible to locate modified peptides by virtue of an alteration to an expected molecular weight (Tab. 12.1). Modified peptides can be chemically/enzymatically treated to remove the modification.
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