Two-dimensional gel electrophoresis in combination with high resolution mass spectrometry, e.g. Fourier Transform-ion cyclotron resonance mass spectrometry (FT-ICR MS) represent efficient techniques which have been established in proteome analysis from biological samples. Muscle proteins have been intensively studied by biochemists in the past focusing their attention mainly on characterization structure and function of these proteins as well as their interaction with other biomolecules. Muscle proteins have been classified into three categories based on their morphological-functional features and extraction by using different buffers as follows: (i), contractile proteins involved in the muscle contraction, (ii), metabolic muscle proteins including enzymes involved in different pathways of muscle energy metabolism, and (iii), structural proteins represented by proteins which maintain the muscle structure. Muscle proteins have attracted wide interest of many researchers because of complex changes in these proteins during myogenesis and cell differentiation followed by formation of variorous types of myofibrils and changes in protein patterns during the development of muscle deseases. Hence, much research effort has been directed into the characterization of muscle structural changes during post-mortem storage with the focus on protein degradation and oxidation. In this work 2-D gel electrophoresis in combination with high resolution FT-ICR mass spectrometry were used as major tools for identification and structural characterization of muscle protein changes. While 2-D gel electrophoresis offers high resolution of protein separation, FT-ICR mass spectrometry provides high resolution, mass accuracy and mass sensitivity leading to unambiguos protein identification.
The first part of the dissertation was concentrated on the mass spectrometric identification of intact and truncated protein products after 2-D gel separation as well as 2-D gel mapping of muscle protein denaturation as a result of pH drop during post-mortem storage. In the initial part a number of different buffers were tested in an attempt to optimize the muscle protein extraction and solubilization. The use of strongly reducing-denaturing buffer yielded a good solubilization of proteins, especially contractile proteins such as actin, tropomyosin and myosin, while the use of non-denaturing solubilization resulted in a good solubilization of metabolic proteins. Proteolytic truncation may result from the action of calpain proteinases
which, although, not directly observed in 2-D gels, due to their very low abundances.
However, MALDI-FTICR MS provided the identification of over 70 intact and truncated protein products. Notable, was the identification of an intact isoform of CKM (mol. weight, 43 kDa) and the identification of two truncation products with molecular weights of 17 and 13 kDa is shown, with sequences corresponding to residues (87-236) and (267-358), respectively, which represent proteolytic cleavage of CKM between residues 237 and 266. The solubilization of muscle proteins in non-denaturing buffer resulted in highly reproducible gels and therefore was used to generate a detailed 2-DE reference protein map for porcine Longissimus dorsi. This map was used to draw a tentative map to examine changes during the post-mortem storage according to the pH change and water loss values and to identify those proteins by mass spectrometry. Muscle proteins were extracted from muscle samples collected 45 min. 24 h and 48 h post-mortem and separated by 2-D gel electrophoresis followed by comparative analysis of the gels using the PDQuest software. 2-D gel maps showed an increase in denaturation of contractile proteins at low pH according to the increase of post-mortem time. Hence, the comparison of low and high pH 24 h M. Semimembranosus showed muscle protein denaturation in both cases. Mass spectrometric analysis of protein gel spots stained with Coomassie-blue showing changes revealed the identification of actin, tropomyosin and several myosin isoforms.
In addition to the Coomassie-blue and silver staining muscle proteins were visualized by a newly developed “stain-free” procedure using the native fluorescence of aromatic amino acids in proteins. In the first series of experiments using a fluorecence Gel Bioanalyser, the sensitivity was tested by comparative analysis of gels run in parallel and stained with Coomassie, silver and native fluorescence detection. The results show that the sensitivity of the stain-free detection is close to silver staining. The fluorescence stability was investigated and it was observed that the fixation of proteins using halogenated derivatives increased the protein stability of prolonged times. Special tools were developed for the exact localization and excision of the protein spots from the gel for subsequent in-gel digestion and mass spectrometric protein identification. A number of proteins such as actin, creatine kinase, triosephosphate isomerase and several isoforms of myosin were successfully identified by MALDI-TOF MS.
The second part of the dissertation was focused on the detection and mass spectrometric identification of muscle protein oxidation, particularly protein carbonylation which may be caused either by direct attack via metal catalysed oxidation to amino acid side chains, or by conjugation with highly reactive carbonyl compounds produced as end-products of lipid peroxidation such as 4-hydroxy-2-nonenal (HNE), malondialdehyde and acrolein. Protein carbonyls were detected by forming a hydrazone derivative with 2,4-dinitrophenylhydrazine (DNP). The derivatized protein products were separated by molecular weight using gel electrophoresis (1-DE and/or 2-DE), blotted to a support matrix (e.g. PVDF), and visualized by immunostaining with antibodies that recognize the DNP portion of the hydrazone. Using this approach a comparative analysis of protein oxidation level between biopsy and 24 h post-mortem was performed. Approximately 70 muscle proteins were detected to be oxidatively modified after gel-immunoblot alignment of which 20 were identified by mass spectrometry. No significant differences of the oxidation levels were observed between the samples therefore further set of experiments was performed by comparing oxidation of protein extracted from muscle with different pH values. In this case the oxidation of myosin light chain 1 and myosin regulatory light chain at high pH was identified.
A further part of this thesis was focused on the identification and mass spectrometric determination of oxidative structure modifications. Such modifications are expressed at very low stoichiometric levels. High performance nano-LC- tandem-mass spectrometry was employed which enabled the identification of several 4-hydroxy-2-nonenal adducts at His and Lys residues of α-actin, creatine kinase and adanylate kinase, which showed high immunoreactivity against anti-DNP antibodies.
No HNE adduct was identified at Cys residue because Cys is usually involved in disulfide bond formation although it is the most nucleophilic candidate for HNE adduct formation. For structure determination collision-induced dissociation (CID) was employed which represents a most widely used peptide fragmentation technique implemented in MS/MS. HNE-containing peptides showed in a neutral loss of HNE (156 Da; 78 or 52 Da for doubly or triply charged peptide) from the precursor or product ions upon CID. Further, mass spectrometric data revealed the identification of Lys oxidation with the formation of aminoadipic semialdehyde.
The third part of the dissertation involved the identification and characterization of physiological protein nitration in sputum of Cystic Fibrosis patients, using a combination of two-dimensional gel electrophoresis, immunoblotting and affinity-mass spectrometric methods. The first experimental step involved sputum protein extraction, separation by 2-D gel and immunoblotting using followed by mass spectrometric identification of protein spots which showed immunoreactvity against anti-3-nitrotyrosine antibodies. The most intense immune response was obtained from leucocyte elastase inhibitor; which was further subjected to nano-LC-tandem MS in an attempt to identified possible tyrosine nitration sites. However, the mass spectrometric data revealed the identification of several hydroxytyrosine structures. Further, an affinity-mass spectrometric experiment was employed in an attempt to enrich the amount of nitrated protein to enable the identification nitration sites. In the initial experiment the immobilization of anti-3-nitrotyrosine antibody on the affinity column was tested using nitrated and non-nitrated proteins prior incubation with sputum proteins. The elution fractions were collected, separated by SDS PAGE and stained with Coomassie blue showing the presence of one consistent band at approximately 80 kDa which was identified as lactotransferrin. All the tandem-MS spectra were revealed the presence of several hydroxytyrosine modifications; however no tyrosine nitration was identified.