Scientific goals of the thesis - Analytical Development and Application of Mass Spectrometry to

1 INTRODUCTION

1.6 Scientific goals of the thesis

Structural and metabolic changes in the muscle during different physiological/pathophysiological conditions as well as structural changes during post-mortem storage have been extensively studied with the focus on the genes and proteins. Proteomic profiling has been applied to define the molecular signature of denervation, immobilization, exercise-training, age-related muscle disease, insulin-resistance, muscular dystrophy and meat quality. Sometimes, determination of structural changes of metabolic enzymes and contractile proteins and their corresponding post-translational modification and/or oxidative post-translational modifications is very difficult, because such structural modifications are present at very low stoichiometrical level. Such problems can be overcome by development of separation techniques coupled with advanced mass spectrometric methods. The major goals of the present thesis were a comprehensive study of muscle proteins structural changes by application of high resolution 2-D gel electrophoresis in combination with high resolution mass spectrometric methods.

The scientific goals of the present dissertation are summarized as follows:

Analytical development and bioanalytical application of high resolution 2-D gel electrophoresis and mass spectrometric methods for identification and structural characterization of meat quality biomarkers: (i), Proteomics analysis of proteome changes during post-mortem storage of muscle samples with different pH values and (ii) structure determination of post-mortem protein degradation products by high resolution MALDI FT-ICR mass spectrometry.

Identification and characterization of oxidation structures occurring in muscle protein by high performance liquid chromatography in combination with collision induced dissociation mass spectrometry: Determination of muscle protein carbonylation sites occurred via (i), lipid peroxidation (ii), metal catalysed oxidation (MCO) and (iii) non-carbonylation protein oxidation

Isolation and separation of sputum proteins extracted from cystic fibrosis patients for identification of physiological protein nitration and oxidation. In this part, a combination of immunologic and affinity-mass spectrometric methods was employed for the identification of specific protein oxidation using specific antibodies.

2 2.1

RESULTS AND DISCUSSION

Mass spectrometric methods for proteome analysis of post-mortem changes of skeletal muscle proteins

2.1.1 Methods of high resolution mass spectrometry for proteome analysis In proteome applications, the various cell proteins are separated by 2-D gels and in-gel digested. MS and MS/MS analyses are used for the protein identification via database searches [23]. In functional proteomics protein complexes containing 2 up to 50 components do not need 2-D gels, but for a safety analysis via SDS-PAGE of small and very large components the analysis of digests of intact isolated complexes is important. Mass spectrometers with high mass resolution and accuracy are taken into consideration for complex protein mixture analysis because of lower requirements for the preceding separation. Proteins can now be analysed by MS to reveal elemental composition, complete or partial amino acid sequence, post-translational modifications, protein-protein interaction sites, and even provide insight into conformational aspects. With these pieces of information new data into the molecular behaviour of individual molecules or molecular ensembles can be obtained directly from mass spectrometric data. As a result, mass spectrometry has evolved into an enabling discipline that plays an increasingly important role in many areas of science, particularly, in characterization of proteins function in many different biological systems.

Of all mass spectrometric methods Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) offers a unique combination of analytical qualities.

An FTICR mass spectrometer combines high resolution, high mass accuracy, non-distructive, multichannel detection, long ion-obsevation times and, most importantly, tools for structural analysis of large molecules. The most important advantages of FT-ICR as a mass analyser is that the ion mass-to-charge ratio is experimentally manifested as a frequency. Because the frequency can be measured more accurately than any other experimental parameter, ICR-MS, offers inherently higher resolution (and thus higher mass accuracy) than any other type of mass measurement. The introduction of FT techniques to ICR MS, by Comisarow and Marshall [35] has brought the advantages of increased speed (factor of 10,000), or increased sensitivity (factor of 100), but also the advantage of fixed magnetic field;

namely, increased mass resolution (factor of 10,000) and increased mass range (factor of 500). Applications that derive from these advantages include determination of chemical formulas, particular in complex mixtures and detection limit in attomole range [20].

However, the high complexity and cost of the FT-ICR, as well as the relatively low space-charge capacities of both analyzers, suggests why new approaches to ion trapping are welcome in tackling the increasingly complex problems in biological mass spectrometry. Therefore, a more compact, less costly, easier to maintain analyzer with comparable performance (for relatively short acquisition time ≤1.8 sec) was desired to supplement the FT-ICR. This technological gap was filled by the LTQ-Orbitrap hybrid mass spectrometer. The coupling liquid chromatography and mass spectrometry improved significantly, the efficiency of proteome analysis by providing sensitivity, resolution and mass accuracy. Hence, the possibility to perform tandem MS experiments (MS/MS) enhanced the capability of structure determination. Despite its relatively recent commercial introduction, the LTQ-Orbitrap has already proven to be an important analytical tool with a wide range of applications. The high resolving power (>150,000) and excellent mass accuracy (specified as ~2–5 ppm, but demonstrated to be as low as 0.2 ppm under favourable conditions) [39] significantly reduce false positive peptide identifications in bottom-up protein analyses. The most important benefit of using LC-MS/MS is the extended dynamic range [40]; complex peptide mixture can be simultaneously analysed containing a wide range of concentrations. These high performance features of the orbitrap can facilitate unambiguous determination of the charge states of fragment ions, as well as identification of oxidative post-translational modifications. The interpretation of LC-MS data is performed routinely in a widely automated fashion as a result of recent development of bioinformatics tools for data acquisition and analysis, although all relevant spectra need to be verified manually.

2.1.2 Protein visualization using native fluorescence and mass spectrometric identification after two-dimensional gel separation

A variety of protein detection and visualisation techniques of protein bands or spots from one and two-dimensional gel electrophoretic separations have been developed and employed in mass spectrometric proteomics. Well established staining procedures for visualization of proteins in gels have used dyes such as Coomassie Brilliant Blue, silver salts and fluorescent dyes (Flamingo, Sypro®Ruby) [16, 97]. While several of these approaches provide high detection sensitivities of proteins, major problems are frequently encountered with the compatibility of staining procedures with the mass spectrometric analysis, background arising from polar staining materials, and the need for applying destaining procedures of isolated proteins. Several procedures have been recently explored in order to overcome these problems, by using unstained gels in gel electrophoretic separations [98, 99].

Fluorescence detection of proteins has been evaluated with pre- or post-electrophoretic incorporation of halogenated compounds such as trichloromethane, trichloroethanol and trichloroacetic acid, which react with tryptophane residues upon treatment with UV light yielding products that show emission in the visible light range suitable for visualization of protein bands [99]. A direct UV fluorescence detection method for unstained proteins in gels was first developed by Roegener et al. who used laser excitation with 280 nm UV light (35 mJ/cm²) and showed the visualisation of proteins in both 1-D and 2-D gel separations with low detection limits (1-5 ng) [98].

A commercial gel bioanalyzer based on native fluorescence has been recently developed (LaVision-BioTec; Bielefeld, Germany) [100]. Native fluorescence detection of proteins was developed in stain-free one and two-dimensional gel electrophoretic separations as a sensitive and efficient approach for mass spectrometric identifications in proteome analysis [18]. Following 1-D or 2-D gel separations, proteins were visualized using sensitive colloidal Coomassie staining and silver staining as described using a GS-800 calibrated imaging densitometer (Bio-Rad, München, Germany), or scanned with the Gel-BioAnalyzer (BAG). The components of the Gel-Bioanalyzer (LaVision-Biotec GmbH, Bielefeld, Germany) are schematically shown in Figure 2.6.

Figure 2.6. Scheme of the Gel-BioAnalyzer (LaVision- Biotec; Bielefeled, Germany), adapted after http://www.lavisionbiotec.com/en/microscopy-products/gelreader/ used for stained free mass spectrometric protein identification

The experimental setup is based on a UV excitation source and a detection system within the UV range. The UV excitation light was generated by a 300 W xenon lamp (265 - 680 nm). The irradiation area was set to 1 cm² at 35 mW/cm² and imaged by three lenses onto a photomultiplier detector. A UV bandpass filter (280-400 nm) is incorporated to block the excitation light from the detection system. From four filter positions (one for UV excitation, three for visible fluorescence), the UV filter transmitting light at λ = 343 ± 65/2 nm was employed. The large reading area (30 x 35 cm2) provided scanning of both 1-D and 2-D gels. The instrument has a removable gel tray and is equipped to read unstained as well as stained protein gels.

In the present study only scanning of unstained gels was applied. High precision polycarbonate tools for localisation and isolation of protein spots were prepared by the Department’s mechanical workshop; after fixation in position on the gel tray, localisation and isolation of gel spots was carried out by moving the gel tray, with positioning and scanning of the gel controlled by the LaVision- Biotec scanning

software. SDS-PAGE separations of several model proteins were initially tested by comparison of Colloidal Coomassie, silver staining, and stain-free detection using native fluorescence and fluorescence upon fixation with halogenated derivatives.

Previously, it has been shown the incorporation of halogenated compounds in polyacrylamide gels either prior to polymerization [99] or subsequent to the electrophoretic separation [101], followed by UV illumination provide fluorescent protein derivatives. However, our model studies showed that protein fixation using halogenated derivatives after PAGE can be omitted, as illustrated by SDS-PAGE separations of lysozyme and myoglobin without fixation (Figure 2.7a), and with fixation (30 min) in 12 % trichloroacetic acid (TCA) (Figure 2.7b).

Figure 2.7. Comparison of native fluorescence detection for 12 % SDS-PAGE separation of 5 µg hen eggwhile lysozyme (lane 1) and 5 µg myoglobin (lane 2) with and without fixation with trichloroacetic acid. For each gel a 5 µl aliquot of molecular weight marker (10– 200 kDa) was used (lane m). (a), no protein fixation was performed; (b), proteins were fixed for 30 min with a 12 % aqueous trichloroacetic acid solution

Upon scanning with the gel bioanalyzer, protein bands were detected with and without fixation, however bands with approximately 30-50 % higher abundance were observed without protein fixation for the model proteins studied. A further increased abundance was obtained by washing the gel band with water which leads to reduced background fluorescence; however, a washing step was found to cause decreased stability of the protein fluorescence. We observed an increased stability (slower decrease) of the fluorescence intensity in proteins within 48 hours after

fixation in 12 % TCA (data not shown), which may be explained by a more stable fluorescence emission of UV-reaction products of tryptophane with TCA. The MALDI-mass spectrometric identification of the gel band of myoglobin isolated from the gel presented in Figure 2.7a (lane 2, without fixation) is shown in Figure 2.8 (see details of protein localisation and isolation below). The band was excised, in-gel digested with trypsin, and the digest mixture analysed by MALDI-TOF-MS, followed by database search employing the MASCOT peptide mass fingerprinting (PMF) search engine. The database search provided unequivocal identification of myoglobin.

Figure 2.8. MALDI-TOF mass spectrum of horse heart myoglobin (lane 2 in Figure 2.7) identified after stain-free gel detection and in-gel digestion with trypsin

For the gel scanned with the Bioanalyzer, no destaining step was required, which provided high sensitivity and considerably lower sample preparation time compared to staining procedures. For the 320 ng protein band, identification was obtained with a score of 83 (64 % sequence coverage). Thus, these model studies clearly showed that gel separation of proteins detected by native fluorescence without a fixation step represents an efficient and sensitive approach for mass spectrometric identification, providing sufficiently fast mass spectrometric analysis of the gel separated proteins.

Comparative gels scanned with the gel Bioanalyzer and stained with Coomassie blue and silver were performed in order to test the sensitivity of the stain-free native fluorescence detection. Corresponding gel separations of mixtures of the two model proteins, immunoglobulin-G and bovine serum albumin at concentrations of 320 - 5 ng/band are compared in Figure 2.9.

Figure 2.9. Sensitivity of stain-free fluorescence detection and visualization in comparison with silver visualization. Protein samples, IgG (150 kDa heavy and light chain dimer) and BSA (67 kDa) were separated in 7 lanes at 320 - 5 ng. Gel areas presented are zoomed regions from 12 % SDS-PAGE separations. (a), silver stained gels; (b), native fluorescence gels

These results indicated comparable sensitivities for the UV fluorescence detection and silver staining with detection limits of approximately 1-5 ng for IgG, while the corresponding Coomassie-stained gel band was not detectable at this concentration (data not shown). The detection limit in the low nanogram range observed is in good agreement with sensitivity data reported by Roegener et al.[98].

In order to evaluate the isolation and localization of protein bands using the stain-free detection, protein extracts from pig muscle were separated and analysed by 2-D gel electrophoresis. Corresponding 12 % separation gels of muscle protein extract were prepared and visualized by native fluorescence and Coomassie staining (Figure 2.10a, b). Using the Bio-Rad PDQuest software approximately 600 µg proteins were detected in the 2-D gel with native fluorescence (Figure 2.10a), while the detection of approximately 350 protein spots was estimated in the Coomassie- stained gel (Figure 2.10b).

Figure 2.10. Protein identification after 2-D gel separation (12 % SDS-PAGE) of a post-mortem porcine muscle sample (1.5 mg total protein per gel). (A) Gel visualized by native fluorescence (B) Gel stained with Coomassie. MALDI-TOF identification of (C) α-actin (spot 1), (D) Creatine kinase (spot 2), (E) Triosephospate isomerase (spot 3), (F) adenylate kinase (spot 4), (G) Myosin regulatory light chain 2 (spot 5)

This suggests comparable detection sensitivity of the protein spots with native fluorescence and Coomassie staining. Since the fluorescence signal depends on the abundance of aromatic amino 12 acids in the protein, proteins with low abundance in aromatic amino acid residues may not be visualized using this method.

Figure 2.10 shows five examples of stained-free gel spots detection using gel Bioanalyser. Following tryptic digestion of isolated gel spots, the MALDI-MS analysis provided the identification of five proteins summarized in Table 2.2.

Table 2.2. Protein identifications in proteome applications using native fluorescence for visualization of gel separated proteins.

Spot Protein Score Peptides

matched Sequence

coverage % Accession number

1 Skeletal α-actin 92 16 70 P68137

2 Creatine kinase M chain 78 22 90 Q5XLD3

3 Triosephosphate isomerase 78 12 28 Q29371

4 Adenylate kinase isoenzyme 1 98 9 49 P00571

5 Myosin regulatory light chain 2 76 9 44 P02608

Using the stain-free gel bioanalyzer enabled the detection and mass spectrometric identification of proteins from gel spots at detection limits in the low nanogram range, similar to silver staining. Moreover, this approach does not require any post-electrophoretic manipulation by destaining and fixation, thus providing advantages for mass spectrometric analysis by reduced background and time needed for sample preparation. The use of fluorescence detection with 2-D gel electrophoresis suggests that this technique can be developed for automated, high-throughput technologies of proteome analysis. Thus, the stain-free fluorescence visualization should be a useful complement to staining techniques of gel electrophoresis for mass spectrometric protein analysis.

2.2 Application of mass spectrometry for identification of post-mortem protein changes of porcine skeletal muscle proteins

2.2.1 Muscle protein changes during post-mortem storage

Multiple factors, including palatability, water-holding capacity, color, nutritional value and safety, determine meat quality. There is a large variation in both the rate and extent of post-mortem tenderization of meat, and this result in the inconsistency of meat tenderness found at the consumer level. It has been known for a long time that meat tenderness improves during cooler storage, and it was suggested almost a century ago that this is due to enzymatic activity [102].

The three factors that determine meat tenderness are background toughness, the toughening phase and the tenderization phase. While the toughening and tenderization phases take place during the post-mortem storage period, background toughness exists at the time of slaughter and does not change during the storage period. The background toughness of meat is defined as ‘‘the resistance to shearing of the unshortened muscle’’ [103], and variation in the background toughness is due to the connective tissue component of muscle. Muscle is made up of many myofibrils. This aspect of the myofibrils is due to the presence of two types of thick and thin filaments which have an order, in that they overlap, forming a repetitive configuration of bands with identical characteristics.

Each myofibril therefore contains a repetitive series of dark and clear bands.

The wide bands of proteins of the muscular fiber, designated A (anisotropic) bands, contain a clear central area, an H zone, which in turn presents a dense M line, while clear bands, called I (isotropic) bands, are each divided in half by a Z line. The distance between two Z lines is known as a sarcomere. Immediately after slaughtering the very well-organized muscle structure begins to desintegrate. A variety of studies have shown that weakening of the myofibers is the key event in tenderization. The most consistently reported ultrastructural change associated with tenderization is breaking at the junction of the I band and Z-disk [104] (Figure 2.11).

Figure 2.11. Major components of muscle sarcomere consist of parallel arrays of ~ 1.0 µm-long thin filaments that integrate with laterally aligned 1,6 µm-long thick filaments. Tropomyosin molecules are associated with each other head to tail forming two polymers per thin filament stabilizing the thin filaments. A sarcomere is defined as the segment between two neighbouring Z-lines (E. Sayas Barbera et. al.)

The degraded proteins are predominantly myofibrillar and structural proteins, which include troponin-I, troponin-T, desmin, vinculin, meta-vinculin, dystrophin, nebulin and titin. Three major cytoskeletal structures are degraded when meat is tender: Z- to Z-line attachments by intermediate filaments, Z- and M-line attachments to the sarcolemma by costameric proteins and the elastic filament protein titin. For several weeks post-mortem little degradation of desmin occurs, and the Z- to Z-line attachments remain largely intact. Detachment of the Z- and M-lines from the sarcolemma is probably not a limiting factor for tenderization. Thus, titin and desmin are likely key substrates that determine meat tenderness [105, 106].

Three proteolytic systems present in muscle have been investigated for their possible role in post-mortem proteolysis and tenderization: the calpain system, the lysosomal cathepsins and the multicatalytic proteinase complex. In addition to being endogenous in skeletal muscle, these proteolytic systems must fulfill two other requirements to consider them involved in post-mortem proteolysis in meat. Firstly, the proteases must have access to the substrates, and secondly, they must be able to reproduce the proteolysis pattern observed after post-mortem storage of meat.

Incubation of myofibrillar proteins with cathepsins results in different degradation

patterns than those that occur during post-mortem storage of muscle and, it is doubtful that cathepsins are released from the lysosomes in post-mortem muscle.

Moreover, the degradation pattern of myofibrillar proteins by multicatalytic proteinase complex does not mimic the degradation pattern observed in post-mortem muscle [107]. This leaves the calpain system or potentially another, not yet investigated, proteolytic system responsible for post-mortem proteolysis of key myofibrillar proteins and the resultant meat tenderization.

Calpains are calcium-activated proteases with an optimum activity at neutral pH. In skeletal muscle, the calpain system consists of at least three proteases, µ-calpain, m-calpain and skeletal muscle-specific µ-calpain, p94 or calpain 3, and an inhibitor of µ- and m-calpain, calpastatin. Both µ- and m-calpain are composed of two subunits with molecular weights of 28 kDa and 80 kDa, respectively [108]. An important characteristic of µ- and m-calpain is that they undergo autolysis in the presence of calcium Calpain 3, a single polypeptide of 94 kDa with sequence homology to the large subunits of µ- and m-calpain [109]. Calpastatin is the endogenous specific inhibitor of µ- and m-calpain. Several isoforms of this protein exist, but the predominant form in skeletal muscle contains four calpain-inhibiting domains. Calpastatin requires calcium to bind, inhibiting calpains and represents also

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