Van Eyk, Dunn - Proteomic and Genomic Analysis of Cardiovascular Disease - 2003 (522919), страница 58
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A comparison of mass spectra recordedbefore and after such treatments can be compared. For example, alkaline phosphatase can be used to remove phosphate groups [39] and PNGase F can be used toremove complete glycan structures from asparagine residues [40]. Sequential removal of sugar residues from N-linked oligosaccharides using a set of endoproteinase enzymes has also been demonstrated [41]. The detection of phosphopeptidesand glycopeptides can be enhanced by the use of product ion scanning [42]. Thesescans are based on the formation of specific signature ions during CID.
Precursorions that give rise to the signature ion of interest are selectively recorded. For example, selection of m/z 204 as the product ion of N-acetylhexosamine, [HexNAc]+,is indicative of glycopeptides [43], whilst selection of m/z 79, the phosphate ionPO3–, may be used to locate phosphopeptides [44]. Alternatively, the loss of phosphoric acid, H3PO4, can be induced from phosphorylated species, which facilitatestheir detection via a Constant Neutral Loss (CNL) experiment [45]. An MS/MSspectrum of a phospho-peptide is shown (Fig. 12.5).
Here, the phospho-peptideoriginates from Neurofilament triplet H (NFH) protein and contains the sequencemotif KSP, which is recognised by a family of proline-dependent protein kinases.A comprehensive analysis of the phosphorylation sites of NFH and other neurofilament proteins has been reported [46].20520612 Mass SpectrometryTab. 12.1 Mass changes of common post-translational modifications of peptides and proteinsModificationMonoisotopic masschangeAverage masschangeHomoserine formed from Met by CNBrPyroglutamic acid from GlnDisulphide bond formationC-terminal amide from GlyDeamidation of Asn and GlnMethylationHydroxylationOxidation of MetFormylationAcetylationCarboxylation of Asp and GluCarboxyamidomethyl (CAM) CysCarboxymethyl (Cmc) CysPhosphorylationSulphationPyridylethyl (PE-Cys)CysteinylationPentoseDeoxyhexoseHexosamineHexoseLipoic acid (amide bond to Lys)N-acetylhexosamineFarnesylationMyristoylationBiotinylation (amide bond to Lys)Pyridoxal phosphate (Schiff base on Lys)PalmitoylationStearoylationGeranylgeranlylationN-acetylneuraminic acid (Sialic acid)GlutathionlyationN-glycolylneuraminic acid5'-Adenosylation4'-PhosphopantetheineADP-ribosylation–29.99281–17.02655–2.01565–0.984020.9840214.0156515.9949115.9949127.9949142.0105643.9898357.0214658.0054879.9663379.95682105.05785119.00410132.04226146.05791161.06881162.05282188.03296203.07937204.18780210.19836226.07760231.02966238.22966266.26096272.25040291.09542305.06816307.09033329.05252339.07797541.06111–30.0935–17.0306–2.0159–0.98470.984714.026915.999415.999428.010442.037344.009857.052058.036779.979980.0642105.1393119.1442132.1161146.1430161.1577162.1424188.3147203.1950204.3556210.3598226.2994231.1449238.4136266.4674272.4741291.2579305.3117307.2573329.2091339.3294541.3052The emergence of proteomics has caused a growing demand for high sensitivityprotein identification and characterisation and the use of mass spectrometry hasbecome a central component of many proteomic research programmes.
Alreadythe emphasis of proteomics is changing from an unfocused high-throughputapproach, where the results are of limited value, to a more focused approach involving detailed analyses of the protein samples. For example, specific proteins ofinterest can be enriched either by cell fractionation methods and/or affinity based12.3 Strategies for Protein and CharacterisationFig. 12.5 A – MS/MS spectrum of a phosphopeptide originating from Neurofilamenttriplet H protein (P12,036; NFH). NFH wasisolated from a 1D gel band reduced, alkylated and digested with trypsin.
Peptides wereseparated using a 75 micron ID PepMap RPcolumn and eluted at a flowrate of 200 nl/min into a Z-spray source fitted to a Q-Tofinstrument. The doubly charged peptide ionat m/z 584.29, relating to Mr 1166.56 Da, wasselected for MS/MS. B – The fragment ionsproduced matched the sequenceTLDVKS*PEAK were S* represents a phosphorylated serine residue. Sample courtesy ofDiane Hanger.protein purification strategies. Typically, the enriched protein sample is then separated by SDS-PAGE and the protein bands characterised by mass spectrometry. Inthis way organelles can be selectively purified and their protein complement determined by mass spectrometry.
Two recent studies on the golgi and nucleolus exemplify this approach [47, 48]. In the case of affinity-based purification methods thetarget protein is either “pulled-down” using a specific antibody or via a moleculartag [21]. Recently, Eaton et al. have targeted proteins which become S-thiolatedduring oxidative stress following ischemia and repurfusion [49]. Here reactive cysteines were labelled with a biotin reagent and purified using streptavidin. Addi-20720812 Mass Spectrometrytionally, if the purification process is carried out under non-denaturing conditions,other proteins associated to the target protein may be pulled down. The N-methylD-aspartate (NMDA) neurotransmitter receptor complex was characterised in thismanner [50].
Here 77 proteins were found to be associated with the complex.Successful purification by affinity based strategies relies on sufficient affinity ofthe protein complex to the bait and on optimised purification steps. To this end,Rigaut and co workers have developed an elegant tandem affinity purification(TAP) strategy [51]. This double tagging approach improves complex recovery andreduces non-specific binding.
Pre-enrichment of a specific protein will also provide more comprehensive sequence coverage, which is necessary for the completecharacterisation of PTM’s or indeed any changes to the published sequences. Forexample, Gatlin et al. recently demonstrated the identification of amino acid sequence variations resulting from single nucleotide polymorphisms (SNPs) by obtaining 99% sequence coverage of human hemoglobin in a single LC/MS/MS experiment [52].At this point we hope the reader will have gained an appreciation of the powerand versatility of mass spectrometry. The exponential growth in the number ofscientific publications in the field of biological mass spectrometry is clear evidence of the tremendous impact the technologies described have had during thelast decade.
We are convinced that the next few years will see many advances inthe technology and also, perhaps more importantly, that mass spectrometry willenable new discoveries and insights into cellular biology and protein function.To end, it is important to note that the results from any mass spectrometry experiment will ultimately depend on the quality of the sample initially. Proteomeresearch requires many scientific disciplines and we believe that the challengesposed can be best met by closely integrating good separation science with state-ofthe-art mass spectrometry and bioinformatics.
Finally, we would like to thank allour colleagues at Proteome Sciences for their assistance and support during thepreparation of this manuscript.12.4ReferencesMacFarlane R. D., Togerson D. F. Californium-252 plasma desorption massspectroscopy.
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