Van Eyk, Dunn - Proteomic and Genomic Analysis of Cardiovascular Disease - 2003 (522919), страница 90
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19.3 Western blots showing the changesin the mole ratio between tropomyosin, TnIand TnT in tissue and myofibrils from rat extensor digital longus (100% fast – sham (S)and fatigued (F), 12% SDS page). Tissue andMF loadings were normalized for actin.While the Tm:actin ratio was the same between tissue and MFs, the actin:TnI and actin:TnT ratios were altered. Western blotting ofthe cytosolic and lipid fractions revealed thepresence of sTnI and sTnT and, to a lesser extent, tropomyosin and actin.mic analysis, there are two main approaches to isolation: traditional myofibril preparations or IN Sequence.
Myofibril isolation was developed specifically to createa fraction of the contractile/structural proteins for use in in vitro functional assaysin order to provide insight into the status of the contractile proteins [52]. IN Sequence is an extraction method we recently developed specifically for striatedmuscle protein that uses buffers and detergents compatible with all protein sepa-32532619 Myofilament Proteomicsration techniques (e.g., 2-DE, HPLC) [34, 53] (Fig.
19.2). The ideal extractionshould both “preserve” the proteome during preparation (i.e., no artifacts or lossof PTMs) and use buffers/detergents compatible with downstream protein separation (i.e., HPLC and 2-DE) and identification methods (western blot, MS).The traditional myofilament (myofibril) preparation is widely used as it providesenzymatically active contractile proteins for in vitro functional assays. In our experience, the mole ratios of the various myofilament proteins are altered during purification. The native thin filament is mainly composed of actin : Tm : Tn at a mole ratioof 7 : 1 : 1 (Tm being a dimer). Purification of myofilament proteins relies on the dif-Fig.
19.4 Representative 1-DE western blotsof myofibrils purified from cultured rabbit cardiac cells (C) probed with an anti-TnI (top)and anti-TnT (bottom) antibody. Albuminfrom the cell culture medium affects the migration of intact TnT. Most of the TnI frag-ments and all of the TnT fragments were lostduring purification of the myofilament proteins used for ATPase assays. Also, the highermolecular weight TnT and TnI products werenot detected.19.3 Proteomic Analysis of the Myofilament Proteinsferent solubilities of the various cellular components, allowing the separation ofmyofibril, cytosolic and lipid (cell membrane) fractions (although sometimes a modified method which only partially removes the membrane proteins is used). Fractionation of myofibrils is imperfect, as various amounts of each myofilament protein arelost to the cytosolic and/or lipid fractions (data not shown).
Critically, during purification, unequal amounts of the different myofilament proteins were lost (Fig. 19.3),even when wash steps were drastically shortened (data not shown). The quantity ofTm and a-actinin present in both the tissue and the isolated myofibrils were similar.However, upon isolation of the myofibrils there was a differential loss in cTnT, cTnI,and cTnC compared to that in the tissue; in other words, the mole ratio between thevarious thin filament proteins was altered following isolation of the myofibrils. Altering the mole ratio of the myofilament proteins can have drastic effects on ATPaseactivity (e.g., co-operativity, basal and maximal activities, and Ca2+ sensitivity; e.g.,[54, 55]).
Furthermore, PTMs can alter protein solubility and their interactionsand affinities with other proteins. Thus, alterations of the myofilament proteomemay exacerbate the change in the mole ratio because of how a modified protein behaves during purification. For example, proteolysis of TnI and TnT has been reportedto occur in both cardiac and skeletal muscle; however, proteolytic fragments of TnIand TnT do not necessarily remain associated with the purified myofibrils (Fig. 19.4).Thus, the consequences of such proteolysis on in vivo function would not be apparent during subsequent biochemical analysis. Ideally, traditional myofilament preparations should allow one to ascribe functional changes to changes in the proteome.
Fig. 19.3 and 19.4 suggest that traditional myofibril preparations can misrepresent the diseased proteome. Therefore, when preservation of the complete myofilament proteome is required (depending on one’s research question), IN-sequenceprovides a compelling and suitable alternative.19.3.2Protein Separation – 2-DEThe myofilament proteome, despite having relatively few (*20) proteins, containsseveral proteins problematic for 2-DE.
These include: a) highly charged proteins(i.e., TnI (pI *9.8) and troponin C (TnC, pI *4.0)); b) large molecular weightproteins (i.e., myosin heavy chain, a-actinin); and c) protein complexes with extremely strong protein-protein interactions (i.e., the Tn complex).
Both cardiac andskeletal TnI and TnT are problematic because of the strong interactions betweenthe troponin subunits, as well as the relative insolubilities of the individual proteins themselves and the number of potential isoforms which may be present(some at very low abundances). Most TnI and TnT isoforms possess nearly identical pIs and molecular weights, requiring very low protein loads and the use ofzoom gels for their resolution. Use of 1M NaCl in the homogenization buffer presumably helps the destruction of inter- and intra-molecular interactions, therebyincreasing the resolution of some of these troublesome proteins as distinct spotswithout streaking [56].
For example, Fig. 19.5 shows the resolution of cardiac andskeletal TnT with and without the addition of 1M NaCl to the homogenization327Fig. 19.5 Western blots showing the effects of the addition of 1MNaCl to the homogenization buffer on cardiac and skeletal TnT andTnI resolution on 2-DE. Resolution of fast skeletal TnI does not re-quire 1M NaCl while cardiac TnI (and slow skeletal TnI – data notshown) does not resolve adequately even though 1M NaCl wasused.32819 Myofilament ProteomicsFig. 19.6 1-DE and 2-DE western blots of skeletal muscle probed withanti-troponin T (A, 4D-11 and JLT-12) and anti-troponin I (B, fast TnI and8B9) antibodies.
The detection of PTMs or isoforms can depend on themAb. mAb 4D-11 detected three isoforms while mAb JLT-12 detected fiveisoforms of skeletal TnI. The detection of a PTM of fast skeletal TnI wasrevealed only by mAb 8B9 (two spots) while mAb Fast TnI detected onlythe unmodified or native (one spot) form.19.3 Proteomic Analysis of the Myofilament Proteins32933019 Myofilament Proteomicsbuffer (final concentration of NaCl in IEF buffer > 25 mM). In the absence of 1MNaCl, substantial streaking of TnT was observed even though other myofilamentproteins (e.g., actin, Tm, and MLC-1 and MLC-2) were resolved. Furthermore, basic proteins were difficult to focus during IEF due to electroendoosmotic effects athigh pH and the resulting cathodic drift within the gel.
Surprisingly, fast skeletalTnI (Fig. 19.6) was suitably resolved while cardiac and slow skeletal TnI were not(Fig. 19.5). Interestingly, these proteins have high amino acid sequence homologybut their behaviors differ radically with solubilization and/or separation by 2-DE.19.3.3Detection, Quantification and IdentificationTo repeat, the challenge in protein detection is to use a method that is 1) compatible with downstream protein identification methods, 2) able to detect all the myofilament proteins, and 3) able to detect both the native and modified forms withina linear range, thereby allowing accurate quantification. Protein visualization ismost often carried out with silver, fluorescent, or coomassie blue stains. Silverstaining is extremely sensitive and is the most common means to detect proteins5 lg10 lgFig.
19.7 A Computer-generated surface blotsof an unidentified protein from silver-stained2-DE gels. Saturation or non-linearity of protein spots on silver-stained 2-DE gel can occur when the protein is present at a high con-20 lgcentration in the sample, but a high load maybe required in order to detect the PTM(s)(top). Therefore, different sample loadings arenecessary to optimize each protein present inthe extract (bottom).19.3 Proteomic Analysis of the Myofilament ProteinsFig.
19.7 B 2-DE silver-stained gel of myofilament-enriched extract from cardiac tissue. Differential abundance of myofilament proteinsrequires multiple protein loads to ensure linearity but still permit detection of all PTMs.for proteomic analysis. The choice of stain represents a tradeoff between sensitivity and linear range. Any disparity in quantity, when present, between the nativeand modified forms of a protein can lead to problems in the detection and quantification of all forms of the protein. This can result in missing a critical protein alteration, as illustrated in Fig.
19.7, which shows an example of a silver-stained 2DE gel with 10 lg of a myofilament-enriched extract from cardiac muscle obtained by IN sequence. While MLC-1 is visualized within the linear range, actin isoverexposed (main peak), leading to its underestimation. Optimal staining of actinwill result in the failure to detect some modified forms of actin, as well as othermyofilament proteins including MLC-1. Ideally, the perfect stain should have thesensitivity of silver with a broad linear range equal or better than that of coomassie blue or fluorescent stains. Fluorescent stains have recently gained attention asthey offer better sensitivity than coomassie blue and have a larger linear range.Their costs, however, have tempered wide acceptance.The key regulatory proteins, the Tn complex, all stain poorly and unevenly witheither silver (especially with non-glutaraldehyde silver staining which is compatible with subsequent MS) or coomassie blue stain, presumably due to the largeclusters of charged amino acid residues throughout their sequences [56].
Themyofilament proteins also stain with silver at different rates as a result of boththeir abundances and abilities to bind silver (data not shown). In cardiac muscle,actin is the first protein observed during silver staining, followed by MLC-1, Tm,MLC-2 and the remaining low abundance proteins. Therefore, it is imperative thateach gel be stained to completion to ensure proper visualization and thus accuratequantification of all proteins on each gel with respect to one another. A series of33133219 Myofilament Proteomicsgels with different protein loads may be required for accurate quantification whenusing silver staining. One interesting aspect of silver staining is that different proteins stain different colors depending on their amino acid composition.
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