Van Eyk, Dunn - Proteomic and Genomic Analysis of Cardiovascular Disease - 2003 (522919), страница 73
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From the masses of the resulting peptides, usually obtained by a MALDI-TOF instrument, a mass map or mass fingerprint of the original protein is obtained [23]. A number of computer programs (e.g. ProteinProspectorTM and MASCOTTM) are available for using the observed peptide masses tosearch gene sequence databases for proteins that fit the mass fingerprint. The proteins can be ranked according to the number of peptide matches; sophisticatedscoring algorithms take the mass accuracy and the percentage of the protein sequence covered into account and calculate a level of confidence for the identification.
Mass fingerprinting when coupled with automated 2DE spot pickers andMALDI-TOF instrumentation has become the primary technology for large-scaleproteomics work.While mass fingerprinting is a very powerful methodology of database searching, it has several important limitations.
Peptide fingerprints are generally sufficient for identification of proteins from completely sequenced genomes; however,splice variants, edited transcripts, significant numbers of post-translational modifications, and other differences leading to unaccounted peptide masses can preventcorrect identification. The most significant limitation of peptide fingerprint baseddatabase searching is the difficulty in assigning the correct identity of proteins incomplex mixture.
Mixtures of three or more proteins increase the level of ambiguity in the mass fingerprint to the extent that usually only the most abundant protein component is identified. More sophisticated search programs are being designed to address this limitation; for example, databases can now be searchediteratively by removing the peptides associated with an unambiguous match [24].MS/MS Identification with Sequence Tags and Primary Amino Acid Sequence. Tandemmass (MS/MS) spectrometric data obtained on peptides from proteins of interestcan also be used to search databases. Because MS/MS spectra contain structuralinformation related to the primary amino acid sequence of the peptide, rather15.2 Proteomic Tools for Smooth Muscle Physiologiststhan just its mass, these searches are generally more specific and discriminating.However, manual evaluation of peptide MS/MS spectra is generally requiredwhich tends to be a tedious and time consuming endevour.
While more laboriousand more difficult to automate than MALDI-TOF analysis, ESI-MS/MS generallyprovides a more reliable method for the positive identification of proteins fromcomplex silver-stained 2D-gels. This is especially true of when multiple proteinsco-migrate within the same 2DE spot and when the database to be searched lacksadequate coverage of the organism”s genome.When protein sequence information is needed to find the gene within the organism, several search approaches exist. The peptide sequence tag method usedpartial sequence interpretation to search databases [25]. Nearly every MS/MS spectrum contains a short “ladder” of fragment ions that are easily interpreted.
Whena few amino acids are combined with the start mass and the end mass of the series, which specify the exact location of the “ladder” sequence in the peptide andthe known cleavage sites of the enzyme. This sequence tag is used to retrieve candidate proteins from the database which have theoretical fragmentation patternsthat match the experimental one. Another method for searching MS/MS data attempts to match the experimental MS/MS spectrum against predicted spectra forall peptides in the database without extracting any sequence information.
SEQUESTTM, a database search tool, performs this function and provides scores in-Fig. 15.2 Strategies for protein identification.A mixture of proteins is first resolved by 2DEand visualized by staining. Individual spotsare excised and subjected to proteolytic digestion. The resulting peptide mixture can beanalyzed either by TOF-MS to determine themass fingerprint or by tandem mass spectro-metry (MS/MS) to determine the amino acidsequence of individual peptides.
Protein identity can be determined by matching the peptide mass fingerprint for TOF-MS data or bydatabase searching with sequence “tags” orwith FASTS (http://fasta.bioch.virginia.edu)for MS/MS data.26126215 Investigations in Smooth Muscle Cell Physiologydicating to what degree the MS/MS spectrum matches the theoretical spectra derived from the database sequence and how differently the next most similar sequence in the database fits the spectrum.If there is no corresponding genome sequence for the organism (or protein) under investigation, then it becomes necessary to identify the protein by comparisonof de novo sequence data with proteins of other organisms in the sequence databases.
A reliable search protocol uses the FASTS search algorithms [26]. FASTSsearches with multiple peptide sequences of unknown order, as obtained by MS/MS-based sequencing, evaluating all possible arrangements of the peptides. Thisprogram is capable of searching peptide sequence information across both DNA(TFASTS) and protein databases (FASTS). The programs can be accessed athttp://fasta.bioch.virginia.edu.15.3Defining Signal Transduction Modules in Smooth MuscleOne area of cardiovascular disease research that has received considerable attention through proteomics is the global characterization of proteins in vascular tissue [27].
Indeed, most proteomic studies of cardiovascular biology have focusedon the construction of 2DE protein databases from cardiomyocytes [28–30]. Determining global changes in protein expression levels of cardiomyocytes in responseto environmental and disease states such as ischemia, hypertension, hypertrophy,infarction, restenosis, atherosclerosis, obesity and diabetes may provide unique insight and understanding of the respective molecular mechanisms of disease.Equally important is the discovery of novel proteins that may play an integralfunction in normal and disease processes. Such identifications may provide basicinformation regarding molecular mechanisms as well as provide targets for noveldrug discovery and biomarkers for a disease state. In comparison to other organs,the heart is a relatively homogenous organ, consisting mainly of myocardial muscle cells, and is therefore well suited for a proteomic investigation by 2DE.
Perhaps for this reason, major initiatives have been highly successful in the development of master myocardial 2DE databases. These myocardial 2DE databases havebeen constructed and are available over the World Wide Web: HSC-2DPAGE [31]and HEART-2D-PAGE [32].
High performance 2DE procedures have resulted inthe resolution of 3,300 myocardial protein species with *300 proteins identified.Studies undertaken to compare healthy versus disease state myocardium haveidentified many non-reproducible spot intensity variations, probably caused by different forms of cardiovascular disease or by parameters including but not limitedto disease stage, medication used, age, gender, and nutritional status. Despite thefact that these studies were undertaken with different sample preparations, different isoelectric focusing conditions, and different gel sizes, the inter-laboratorycomparison has shown that spots identified by sequencing methods appeared atthe same positions and identification by pattern comparison was successful inmany cases.
The continued refinement of 2DE technology, bioinformatics and MS15.3 Defining Signal Transduction Modules in Smooth Musclesequencing sensitivity should continue to foster advancements in the cardiovascular expression proteomics.Rather than use protein expression proteomics to simply characterize the largenumber of proteins present in SMC, we recognized the true power of this technology in its ability to enhance the output of existing approaches currently used by themodern physiologist. We prefer to let the biological question drive the application ofproteomics.
Thus, rather than define an entire cellular proteome (a laborious process that is perhaps beyond the scope of an academic lab [33]) we have defined experiments to select a number of relevant proteins in the mixture of interest. Examples of such experiments that we routinely perform are the definition of early phosphorylation events after agonist treatment of intact SMC, and the comparison of protein complex components in affinity pull down experiments from stimulated andnon-stimulated SMC. Only proteins that are demonstrated to be specifically phosphorylated or associated with a protein complex in response to the stimulus are examined. Thus, we can eliminate a great deal of extraneous information by sequencing only those proteins that are demonstrated to be regulated; these select proteintargets then direct all subsequent biological investigations.Recent work in our laboratory has been using a proteomic approach to analyzecalcium sensitization and desensitization in smooth muscle and vascular tissue.The major mechanism for adjusting the sensitivity of the contractile apparatus insmooth muscle is through the sensitization of the 20-kDa myosin light chain(MLC20) phosphorylation and contractile force to [Ca2+]i [34–36].
However, stimulation of contraction can be induced with several agonists at submaximal, fixedCa2+ levels [37]. This regulation of force and MLC20 phosphorylation that is independent of changes in [Ca2+]i is referred to as calcium sensitivity [37, 38]. The extent of MLC20 phosphorylation depends on the relative activities of myosin lightchain kinase (MLCK) and myosin light chain phosphatase (SMPP-1M). “Ca2+-sensitization” is chiefly mediated via a G-protein linked pathway that acts to inhibitSMPP-1M activity [39, 40].
SMPP-1M is composed of three subunits: the 37 kDacatalytic subunit of PP-1 (PP-1C); a 110–130 kDa regulatory myosin phosphatasetargeting subunit (MYPT1) and a 20 kDa subunit of undetermined function [41].The myosin phosphatase activity of SMPP-1M is known to be regulated by phosphorylation of the MYPT1 subunit at an inhibitory site of phosphorylation by anunidentified endogenous kinase [42].To identify the relevant endogenous kinase responsible for the inhibitory phosphorylation of SMPP-1M, we used the “purine binding cassette sub-proteome” isolated from bladder tissue. The most critical stage of any functional proteomicsapproach is the strategic design for the isolation of protein targets.
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