Proteomic changes in hearts of kyphoscoliosis (ky) mutant mice in the absence of structural pathology: implication for the analysis of early human heart disease

Complex molecular changes associated with early stage human heart disease are poorly understood and prevent the development of effective treatments of human cardiac disease. Relatively minor structural changes in early disease may accompany some conditions such as arrhythmias. Our objective was to determine if significant proteomic changes occur in heart tissues in the absence of structural pathology. We used a proteomic "pipeline" based on Ciphergen SELDI-TOF/MS, gel electrophoresis and MALDI-TOF/MS. The kyphoscoliosis (ky) mouse carries a mutation in a putative transglutaminase causing a primary skeletal muscle disease. The ky protein is expressed usually in skeletal and cardiac muscle but its absence from the ky heart causes no structural pathology making it a good model of "occult" heart disease. We discovered 20 statistically validated biomarkers discriminating ky from normal hearts, one cardiac troponin-I was reduced by 40% in ky hearts. A 17% deficit was confirmed subsequently by Western blot. Thus, the proteome of ky hearts was abnormal, giving support to our contention that this SELDI-based analytical approach is capable of making a significant contribution to the analysis of complex proteomic changes in early stage human heart disease.     

Comparative proteomic analysis of the contractile-protein-depleted fraction from normal versus dystrophic skeletal muscle

In basic and applied myology, gel-based proteomics is routinely used for studying global changes in the protein constellation of contractile fibers during myogenesis, physiological adaptations, neuromuscular degeneration, and the natural aging process. Since the main proteins of the actomyosin apparatus and its auxiliary sarcomeric components often negate weak signals from minor muscle proteins during proteomic investigations, we have here evaluated whether a simple prefractionation step can be employed to eliminate certain aspects of this analytical obstacle. To remove a large portion of highly abundant contractile proteins from skeletal muscle homogenates without the usage of major manipulative steps, differential centrifugation was used to decisively reduce the sample complexity of crude muscle tissue extracts. The resulting protein fraction was separated by two-dimensional gel electrophoresis, and 2D-landmark proteins were identified by mass spectrometry. To evaluate the suitability of the contractile-protein-depleted fraction for comparative proteomics, normal versus dystrophic muscle preparations were examined. The mass spectrometric analysis of differentially expressed proteins, as determined by fluorescence difference in-gel electrophoresis, identified 10 protein species in dystrophic mdx hindlimb muscles. Interesting new biomarker candidates included Hsp70, transferrin, and ferritin, whereby their altered concentration levels in dystrophin-deficient muscle were confirmed by immunoblotting.     

Proteomic analysis of rat soleus and tibialis anterior muscle following immobilization

A proteomic analysis was performed comparing normal slow twitch type fiber rat soleus muscle and normal fast twitch type fiber tibialis anterior muscle to immobilized soleus and tibialis anterior muscles at 0.5, 1, 2, 4, 6, 8 and 10 days post immobilization. Muscle mass measurements demonstrate mass changes throughout the period of immobilization. Proteomic analysis of normal and atrophied soleus muscle demonstrated statistically significant changes in the relative levels of 17 proteins. Proteomic analysis of normal and atrophied tibialis anterior muscle demonstrated statistically significant changes in the relative levels of 45 proteins. Protein identification using mass spectrometry was attempted for all differentially regulated proteins from both soleus and tibialis anterior muscles. Four differentially regulated soleus proteins and six differentially regulated tibialis anterior proteins were identified. The identified proteins can be grouped according to function as metabolic proteins, chaperone proteins, and contractile apparatus proteins. Together these data demonstrate that coordinated temporally regulated changes in the proteome occur during immobilization-induced atrophy in both slow twitch and fast twitch fiber type skeletal muscle.     

Proteomic analysis of bovine skeletal muscle hypertrophy

Myostatin plays a major role in muscle growth and development and animals with disruption of this gene display marked increases in muscle mass. Little is known about muscle physiological adaptations in relation to this muscle hypertrophy. To provide a more comprehensive view, we analyzed bovine muscles from control, heterozygote and homozygote young Belgian blue bulls for myostatin deletion, which results in a normal level of inactive myostatin. Heterozygote and homozygote animals were characterized by a higher proportion of fast-twitch glycolytic fibers in Semitendinosus muscle. Differential proteomic analysis of this muscle was performed using two-dimensional gel electrophoresis followed by mass spectrometry. Thirteen proteins, corresponding to 28 protein spots, were significantly altered in response to the myostatin deletion. The observed changes in protein expression are consistent with an increased fast muscle phenotype, suggesting that myostatin negatively controls mainly fast-twitch glycolytic fiber number. Finally, we demonstrated that differential mRNA splicing of fast troponin T is altered by the loss of myostatin function. The structure of mutually exclusive exon 16 appears predominantly expressed in muscles from heterozygote and homozygote animals. This suggests a role for exon 16 of fast troponin T in the physiological adaptation of the fast muscle phenotype.     

Relative quantification in proteomics: new approaches for biochemistry

Recent developments in mass spectrometry and protein arrays provide opportunities to derive systematically proteomic information from small samples of cellular material. Relative quantification among samples can be achieved with either gel-based or gel-free approaches. Furthermore, the adaptation of specific techniques facilitates absolute quantification. Here, relative quantification in two-dimensional gel electrophoresis is contrasted with that in non-gel-based approaches such as isobaric tagging of peptides, pre-labelling of living cells with isotopomeric forms of essential amino acids and protein array platforms. In addition, novel flow-cytometry-based approaches are considered. These technologies can all be used to determine accurately the levels of proteins or biomarkers in a wide range of samples.     

Establishment of a near-standard two-dimensional human urine proteomic map

A proteomic map for human urine on two-dimensional (2-D) gels has been developed. Initial studies demonstrated that the urine proteins prepared by conventional methods showed interference and poor reproducibility in 2-D electrophoresis (2-DE). To address this issue, urine samples were dialyzed to remove any interfering molecules. The dialysis of urine proteins and the concentration by lyophilization without fractionation significantly improved the reproducibility and resolution and likely represents the total urine proteins on a 2-D gel. In addition, removing albumin from urine using Affi-Gel Blue helped to identify the low-abundant proteins. Using the developed method, we prepared proteins from urine collected from healthy females and males. The large inter- and intra-subject variation in protein profiles on 2-D gels made it difficult to establish a normal human urine proteomic 2-D map. To resolve this problem, urinary proteins were prepared from the pooled urine collected from 20 healthy females and males, respectively. The established male and female urine proteomes separated on 2-D gels were almost identical except for some potential sex-dependent protein spots. We have annotated 113 different proteins on the 2-D gel by peptide mass fingerprinting (PMF). We propose that the established total urine proteome can be used for 2-DE analysis, liquid chromatography-tandem mass spectrometry (LC-MS/MS), and identification of novel disease-specific biomarkers.     

Regulation of cell proliferation by fast Myosin light chain 1 in myoblasts derived from extraocular muscle, diaphragm and gastrocnemius

The extraocular muscle (EOM) suffers much less injury from Duchenne muscular dystrophy (DMD) than other skeletal muscles such as diaphragm and gastrocnemius. The present study was undertaken to test the hypothesis that differential expression of regulatory proteins between the EOM and other skeletal muscles is responsible for the observed difference in the sensitivity to DMD-associated damage. Protein expression in the tissue samples obtained from EOM, diaphragm or gastrocnemius of C57BL/6 mice was analyzed by two-dimensional gel electrophoresis and mass spectrometry. There were 35 proteins that were identified to be differentially expressed among different skeletal muscle tissues. Among the 35 proteins, a fast skeletal muscle isoform myosin light chain 1 (MLC1f) protein was further studied in relation to muscle cell proliferation. The EOM-derived myoblasts had much lower levels of MLC1f and higher rate of cell proliferation in contrast to the myoblasts derived from diaphragm or gastrocnemius, which displayed a higher expression of MLC1f along with a slow proliferation. Deletion of MLC1f using siRNA targeting MLC1f resulted in an increased rate of cell proliferation in the myoblasts. Cell cycle analysis revealed that MLC1f inhibited the transition of the cell cycle from the G1 to the S phase. Therefore, the present study demonstrates that MLC1f may negatively regulate proliferation of myoblasts through inhibition of the transition from the G1 to the S phase of the cell cycle. Low levels of MLC1f in myoblasts of EOM may ensure cell proliferation and enhance the repair process for EOM under the DMD disease condition, thus making EOM suffer less injury from DMD.     

Skeletal muscle proteomics: carbohydrate metabolism oscillates with seasonal and torpor-arousal physiology of hibernation

The physiology of small mammalian hibernators shifts profoundly over a year, from summer homeothermy to winter heterothermy. Torpor-arousal cycles define high-amplitude tissue activity fluctuations in winter, particularly for skeletal muscle, which contributes to the energetically demanding rewarming process via shivering. To better understand the biochemistry underlying summer-winter and torpor-arousal transitions, we applied two-dimensional gel electrophoresis coupled with liquid chromatography/mass spectrometry/mas spectrometry to the soluble proteins from hindlimb muscle of 13-lined ground squirrels (Ictidomys tridecemlineatus) in two summer and six winter states. Two hundred sixteen protein spots differed by sampled state. Significantly, intrawinter protein adjustment was a minor component of the dataset despite large discrepancies in muscle activity level among winter states; rather, the bulk of differences (127/138 unequivocally identified proteins spots) occurred between summer and winter. We did not detect any proteomic signatures of skeletal muscle atrophy in this hibernator nor any differential seasonal regulation of protein metabolism. Instead, adjustments to metabolic substrate preferences dominated the detected proteomic differences. Pathways of carbohydrate metabolism (glycolysis and gluconeogenesis) were summer enriched, whereas the winter proteome was enriched for fatty acid β-oxidation. Nevertheless, our data suggest that some reliance on carbohydrate reserves is maintained during winter. Phosphoglucomutase (PGM1), which reversibly prepares glucose subunits for either glycolysis or glycogenesis, showed apparent winter state-specific phosphorylation. PGM1 was phosphorylated during rewarming and dephosphorylated by interbout arousal, implying that glucose supplements lipid fuels during rewarming. This, along with winter elevation of TCA cycle enzymes, suggests that hindlimb muscles are primed for rapid energy production and that carbohydrates are an important fuel for shivering thermogenesis.     

Serum protein profiling to identify biomarkers for small renal cell carcinoma

Diagnostic biomarkers for early detection of renal cell carcinoma (RCC) are in great need. In the present study, we compared the serum protein profiles of patients with small RCC to those of healthy individuals to identify the differentially expressed proteins with potential to serve as biomarkers. Serum samples were collected from 10 patients with small RCC and 10 healthy individuals. The serum protein expression profiles were analyzed by two-dimensional (2-D) gel electrophoresis. Twenty-seven proteins with differences in expression levels between RCC patients and healthy volunteers were identified. Of these, 19 were expressed at different levels and eight were expressed in serum from the RCC group, but not from the control group. Six differentially expressed proteins identified by using mass spectrometry included coagulation factor XIII B, complement C3 and its precursor, misato homolog 1 (isoform CRA_b), hemopexin, and alpha-1-B-glycoprotein. Some of these serum proteins are known regulators of tumor progression in human malignancies. In conclusion, we successfully applied 2-D gel electrophoresis and identified six serum proteins differentially expressed between patients with small RCC and healthy volunteers. These proteins may provide novel biomarkers for early detection and diagnosis of human RCC.     

Developmental changes of cardiac and slow skeletal muscle troponin T expression in chicken cardiac and skeletal muscles

Numerous troponin T (TnT) isoforms are produced by alternative splicing from three genes characteristic of cardiac, fast skeletal, and slow skeletal muscles. Apart from the developmental transition of fast skeletal muscle TnT isoforms, switching of TnT expression during muscle development is poorly understood. In this study, we investigated precisely and comprehensively developmental changes in chicken cardiac and slow skeletal muscle TnT isoforms by two-dimensional gel electrophoresis and immunoblotting with specific antisera. Four major isoforms composed of two each of higher and lower molecular weights were found in cardiac TnT (cTnT). Expression of cTnT changed from high- to low-molecular-weight isoforms during cardiac muscle development. On the other hand, such a transition was not found and only high-molecular-weight isoforms were expressed in the early stages of chicken skeletal muscle development. Two major and three minor isoforms of slow skeletal muscle TnT (sTnT), three of which were newly found in this study, were expressed in chicken skeletal muscles. The major sTnT isoforms were commonly detected throughout development in slow and mixed skeletal muscles, and at developmental stages until hatching-out in fast skeletal muscles. The expression of minor sTnT isoforms varied from muscle to muscle and during development.     

Comparative proteomic analysis of hearts of adult SCNT Bama miniature pigs (Sus scrofa)

This study aims to determine the effects of SCNT on cardiac development of SCNT pigs through proteomic methods. Heart proteins from three adult SCNTs and two normal reproductive Bama miniature pigs were extracted, separated, and identified via comparative proteomic methods, including two-dimensional gel electrophoresis, mass spectrometry, and Western blot. Eleven differentially expressed spots were identified as differentially expressed proteins, of which five spots were upregulated proteins such as cardiac myosin heavy chain, cathepsin D, and heat shock protein beta-1 (HSP27). By contrast, six spots were downregulated proteins such as alpha skeletal muscle and actin. The results also demonstrated that nuclear transfer might result in abnormal expression of some important proteins in hearts from SCNT pigs, and affect the cardiac development in SCNT pigs' survival.     

Identification of avocado (Persea americana) root proteins induced by infection with the oomycete Phytophthora cinnamomi using a proteomic approach

Avocado root rot, caused by Phytophthora cinnamomi, is the most important disease that limits avocado production. A proteomic approach was employed to identify proteins that are upregulated by infection with P. cinnamomi. Different proteins were shown to be differentially expressed after challenge with the pathogen by two-dimensional (2-D) gel electrophoresis. A densitometric evaluation of protein expression indicated differential regulation during the time-course analyzed. Some proteins induced in response to the infection were identified by standard peptide mass fingerprinting using matrix-assisted laser desorption/ionization-time of flight-mass spectrometry and sequencing by MALDI LIFT-TOF/TOF tandem mass spectrometry. Of the 400 protein spots detected on 2-D gels, 21 seemed to change in abundance by 3 hours after infection. Sixteen proteins were upregulated, 5 of these were only detected in infected roots and 11 showed an increased abundance. Among the differentially expressed proteins identified are homologs to isoflavone reductase, glutathione S-transferase, several abscisic acid stress-ripening proteins, cinnamyl alcohol dehydrogenase, cinnamoyl-CoA reductase, cysteine synthase and quinone reductase. A 17.3-kDa small heat-shock protein and a glycine-rich RNA-binding protein were identified as downregulated. Our group is the first to report on gene induction in response to oomycete infection in roots from avocado, using proteomic techniques.     

Differential expression of equine muscle biopsy proteins during normal training and intensified training in young standardbred horses using proteomics technology

The major aim of the present study was to investigate the proteome of standardbred horses at different stages of training and intensified training. We searched for biomarkers using small skeletal muscle biopsies of live animals. 2D gel electrophoresis and mass spectrometry were successfully applied to investigate training-induced differential expression of equine muscle biopsy proteins. Despite the poor resolution of the equine genome and proteome, we were able to identify the proteins of 20 differential spots representing 16 different proteins. Evaluation of those proteins complies with adaptation of the skeletal muscle after normal training involving structural changes towards a higher oxidative capacity, an increased capacity to take up long-chain fatty acids, and to store energy in the form of glycogen. Intensified training leads to additional changed spots. Alpha-1-antitrypsin was found increased after intensified training but not after normal training. This protein may thus be considered as a marker for overtraining in horses and also linked to overtraining in human athletes.     

Drastic increase of myosin light chain MLC-2 in senescent skeletal muscle indicates fast-to-slow fibre transition in sarcopenia of old age

The age-dependent decline in skeletal muscle mass and function is believed to be due to a multi-factorial pathology and represents a major factor that blocks healthy aging by increasing physical disability, frailty and loss of independence in the elderly. This study has focused on the comparative proteomic analysis of contractile elements and revealed that the most striking age-related changes seem to occur in the protein family representing myosin light chains (MLCs). Comparative screening of total muscle extracts suggests a fast-to-slow transition in the aged MLC population. The mass spectrometric analysis of the myofibril-enriched fraction identified the MLC2 isoform of the slow-type MLC as the contractile protein with the most drastically changed expression during aging. Immunoblotting confirmed an increased abundance of slow MLC2, concomitant with a switch in fast versus slow myosin heavy chains. Staining of two-dimensional gels of crude extracts with the phospho-specific fluorescent dye ProQ-Diamond identified the increased MLC2 spot as a muscle protein with a drastically enhanced phosphorylation level in aged fibres. Comparative immunofluorescence microscopy, using antibodies to fast and slow myosin isoforms, confirmed a fast-to-slow transformation process during muscle aging. Interestingly, the dramatic increase in slow MLC2 expression was restricted to individual senescent fibres. These findings agree with the idea that aged skeletal muscles undergo a shift to more aerobic-oxidative metabolism in a slower-twitching fibre population and suggest the slow MLC2 isoform as a potential biomarker for fibre type shifting in sarcopenia of old age.     

Analysis of complex protein-polypeptide systems for proteomic studies

Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), followed by protein extraction and characterization with chemical sequencing or mass spectrometry (MS), is the most commonly used method to analyze complex protein systems such as cells and organelles. However, it is claimed that 2-D PAGE is a slow and labor-intensive technique and also needs subsequent efforts for one-by-one identification of proteins. Recently, the combined methods of Fourier transform ion cyclotron resonance (FTICR) mass spectrometry, with preceding separation techniques such as capillary isoelectric focusing (CIEF) or liquid chromatography, have been demonstrated as high-throughput techniques suitable for proteomic analysis of protein systems. The studies which employ FTICR MS, aimed at the analysis of complex protein systems, have been reviewed, comparing their performance with that of 2-D PAGE. Also, the possibilities of combining 2-D PAGE and the FTICR MS method to analyze and reconstruct the structures and functions of complex systems are discussed.     

Proteomic analysis of secreted muscle components: search for factors involved in neuromuscular synapse formation

Denervated but not innervated skeletal muscles secrete polypeptides that are involved in neuromuscular synapse formation. With the aim of identifying such components, metabolically labeled polypeptides in extracts from denervated and innervated muscles were submitted to two-dimensional gel electrophoresis, and the abundance of individual molecular species was compared. Consistent differences between the proteomic maps from the two sources of muscles were seen. Likewise, proteomic maps of polypeptides from organ culture media conditioned by chronically denervated muscles and by control muscles revealed consistent differences, but the abundance of material within individual spots from conditioned media was not sufficient for analysis by mass spectrometry. Since it was not possible to match the patterns from muscle extracts and from conditioned media, it has been established that extract of Sol8 muscle cells was a satisfactory source of material for analysis. From 1,200 spots identified on the proteomic map from Sol8 cells by image analysis, some 140 have been defined by mass spectrometric analysis. In order to identify the components that are shared by secreted molecules from denervated muscles and Sol8 cells, a mixture of extracts from the two sources was co-electrophoresed and a shared proteomic pattern was established by visualization of metabolically labeled spots from the conditioned medium and of silver stained spots from the Sol8 cells. More than 100 spots sharing x/y coordinate localization could be seen on the pattern. Of these, fourteen were among those identified by mass spectrometry. It is concluded that co-electrophoresis of radioactively labeled polypeptides from conditioned media with extracts from Sol8 cells can be used to mark in the proteome of Sol8 cells those polypeptides that are secreted at low abundance by adult muscles. Their higher abundance in Sol8 cells opens the possibility for further scrutiny of spots by mass spectrometry or by microsequencing.     

Contractile proteins of fast and slow fibers during differentiation of lobster claw muscle

Contractile protein populations were determined, using gel electrophoresis, during development of the claw closer muscles of the lobster Homarus americanus. In the adult the paired claw closer muscles are asymmetric, consisting of a crusher muscle with all slow fibers and a cutter muscle with a majority of fast and a few slow fibers. The electrophoretic banding pattern of these adult fast and slow fibers shows a similarity in the major proteins including myosin, actin, and tropomyosin which are common to both fiber types. Paramyosin is slightly heavier in fast fibers than in slow. However, fast fibers have three proteins and slow fibers have four proteins which are unique to themselves. Several of these unique proteins belong to the regulatory troponin complexes. In juvenile 4th stage lobster, where the paired closer muscles are undifferentiated, the banding pattern reveals the presence of proteins common to both fiber types including myosin, actin, and tropomysin but the conspicuous absence of all unique fast fiber proteins as well as one unique slow fiber protein. By the juvenile 10th stage most of these unique proteins are present except for one unique slow fiber protein. Thus lobster fast and slow fiber differentiation entails coordinate gene activation to add unique contractile proteins.     

Immunochemical comparison of myosin light chains from chicken fast white, slow red, and cardiac muscle.

Antibodies were formed against the myosin light chains isolated from chicken fast skeletal, slow skeletal, and cardiac muscle and the antigenicities of the light chains were compared by double immunodiffusion and immunoelectrophoresis. It was shown that fast light chains are immunologically different from light chains of slow and cardiac myosin, while the slow and cardiac muscle light chains have similar immunological characteristics; that is, the light chains of apparent molecular weight about 27,000 daltons in SDS-acrylamide gel electrophoresis of slow and cardiac muscle are immunologically indistinguishable, and the other light chains of apparent molecular weight about 19,000 daltons of both muscles include a common antigenic site.