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Originally published In Press as doi:10.1074/mcp.M600345-MCP200 on January 16, 2007.
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Molecular & Cellular Proteomics 6:728-737, 2007.
© 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

Research

Quantitative Proteomics Profiling of Sarcomere Associated Proteins in Limb and Extraocular Muscle Allotypes*,S

Sven Fraterman{ddagger},§,¶,||, Ulrike Zeiger**,¶, Tejvir S. Khurana**,¶, Matthias Wilm{ddagger} and Neal A. Rubinstein§,¶

From the {ddagger} Gene Expression Unit, European Molecular Biology Laboratory, 69117 Heidelberg, Germany and § Department of Cell and Developmental Biology, ** Department of Physiology, and Pennsylvania Muscle Institute, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The sarcomere is the major structural and functional unit of striated muscle. Approximately 65 different proteins have been associated with the sarcomere, and their exact composition defines the speed, endurance, and biology of each individual muscle. Past analyses relied heavily on electrophoretic and immunohistochemical techniques, which only allow the analysis of a small fraction of proteins at a time. Here we introduce a quantitative label-free, shotgun proteomics approach to differentially quantitate sarcomeric proteins from microgram quantities of muscle tissue in a fast and reliable manner by liquid chromatography and mass spectrometry. The high sequence similarity of some sarcomeric proteins poses a problem for shotgun proteomics because of limitations in subsequent database search algorithms in the exclusive assignment of peptides to specific isoforms. Therefore multiple sequence alignments were generated to improve the identification of isoform specific peptides. This methodology was used to compare the sarcomeric proteome of the extraocular muscle allotype to limb muscle. Extraocular muscles are a unique group of highly specialized muscles with distinct biochemical, physiological, and pathological properties. We were able to quantitate 40 sarcomeric proteins; although the basic sarcomeric proteins in extraocular muscle are similar to those in limb muscle, key proteins stabilizing the connection of the Z-bands to thin filaments and the costamere are augmented in extraocular muscle and may represent an adaptation to the eccentric contractions known to normally occur during eye movements. Furthermore, a number of changes are seen that closely relate to the unique nature of extraocular muscle.

The sarcomere is the basic functional unit of striated muscle. Its major components are the actin-rich thin filaments attached to the lateral Z-bands ("Zwischenscheiben") and the medial M-bands ("Mittelscheibe") cross-linking the myosin containing thick filaments. Although the existence of these compartments and their component proteins have been known for a long time, several new protein classes have been identified with the sarcomere in the last decade and established it as a dynamic and complex protein network defining and regulating the properties of different muscle allotypes under various conditions (1–3).

Although cardiac and skeletal muscle remain the two major classes of striated muscle, several specialized sub classes or "allotypes" have been identified in the cranio-facial region (4–7). Of these EOM1 are the best characterized example of such a highly adapted muscle allotype (5, 8–14). A unique characteristic of EOM is their compartmentalization into two distinct layers, the orbital layer and global layer. Their functional requirements range from slow vestibulo-ocular and optokinetic eye reflexes to rapid saccadic eye movements exceeding 600°/s (15). They can generate twitch contractions at 400 Hz without tetanus and undergo extensive eccentric contractions, which are unusual during normal limb muscle movements. Although the exact mechanisms by which EOM attain this unique set of functional properties are not yet fully understood, diversification of sarcomeric proteins along the muscle is thought to play an important role. This is exemplified by expression of EOM-specific MYH13 at the central endplate region but slower embryonic MYH3 at the ends of myofibers (12, 16). From a disease perspective, they are preferentially involved in myasthenia gravis and some mitochondrial myopathies, although they are spared in Duchenne muscular dystrophy.

Although advances in microarray technology have begun to allow insight into the diversity of sarcomeres in muscle allotypes (9) or disease (17) on the mRNA level, the analysis of sarcomere associated proteins has relied on targeted but low complexity approaches like immunohistochemistry (12) and one-dimensional gel electrophoresis based techniques (14, 18). Proteomics have already enabled several studies on muscle tissue using two-dimensional gel electrophoresis and subsequent mass spectrometric identification (19, 20). Because the sarcomere is a repetitive structure containing a large number of highly abundant proteins, we propose a shotgun approach in combination with label-free quantitation. Shotgun proteomics uses the sequencing capabilities of modern mass spectrometers to identify proteins based on their HPLC separated tryptic peptides (21). These peptides can also be used to determine the relative peptide and subsequent protein amounts in two or more samples based on their ion intensity over time. Compared with isotope-labeling techniques (22, 23), label-free approaches have the advantage of not requiring special sample preparation, and a protein needs to be identified in only a single sample to be quantitated by its peptides in other samples later (24, 25). The high sequence identity of numerous protein families of the sarcomere poses a problem to shotgun proteomics because of overlapping peptides, which can falsify the quantitation result. Therefore the accurate quantitation of different MYH isoforms and other closely related proteins depends on a strategy that identifies isoform specific peptides (26).

The presented approach is used to analyze the sarcomeric proteome from EOM in relation to the proteome of a fast limb muscle, the EDL. To assess the significance of these results, we compared the protein ratios to the changes in mRNA expression levels from previous studies (5, 8, 9, 11, 27) and validated key changes by immunohistochemistry and Western blot analysis.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
All chemicals were purchased from Sigma unless stated otherwise. Graphs were prepared using Microsoft Excel (Microsoft, Redmond, WA) or GraphPad Prism (San Diego, CA).

Preparation of Rat Muscle Tissue—
Animals were used in accordance with principles in the care and use of animals of the University of Pennsylvania. Three adult female Sprague-Dawley rats were sacrificed by CO2 inhalation. Using sterile conditions both EDL from distal hind limbs and EOM were isolated and kept on ice. Muscles were washed three times in ice-cold PBS prior to transfer to 200 µl of 5 mM PBS and 2% proteinase inhibitor mixture. The two samples were ultrasonicated for 5 min on ice, and then 200 µl of trifluoroethanol was added and incubated for 1 h at 60 °C while shaking. After another 2 min of ultrasonication on ice, the samples were lyophilized and stored at –80 °C until further usage (28).

For analysis, 2 mg of lysate (corresponding to 6 mg of wet muscle tissue) was re-dissolved in 30 mM ammonium bicarbonate and ultrasonicated for 2 min. The lysates were reduced in 200 mM DTT, alkylated in 500 mM iodacetamide, and digested with sequencing grade trypsin (Roche Applied Science) overnight at 37 °C in a total volume of 200 µl. After digestion, the lysate was cleared at 5000 rpm for 5 min, and 10 µl (corresponding to 300 µg of wet muscle tissue) was purified by a µ-C18 ZipTip (Millipore, Schwalbach, Germany).

Capillary LC-MS/MS Analysis—
The samples were separated on a nano-flow 1D-plus Eksigent (Eksigent, Dublin, CA) HPLC system coupled to a qStar Pulsar i quadrupole time-of-flight MS (Applied Biosystems, Darmstadt, Germany).

The digest was loaded onto a 100-µm inner diameter fused silica CapRod monolithic C18 pre-column and washed with Phase A (2% acetonitrile and 0.5% acetic acid in water) (all Merck, Darmstadt, Germany). The reverse-phase separation was performed on a column made by slurry packing 3 µm of YMC C18 particles (YMC, Dinslaken, Germany) into a tapered 20-cm 100-µm inner diameter fused silica capillary (Optronis, Kehl, Germany). The peptides derived from muscle samples were separated by a linear gradient that started at 100% mobile phase A and increased the mobile phase composition to 50% B (0.5% acetic acid in 98% acetonitrile) over a span of 120 min at a constant flow rate of 200 nl/min. Each run was followed by 30 min at 100% mobile phase B. Peptides derived from digested standard proteins were separated by a similar gradient over 30 min. The MS was operated in data-dependent mode. MS spectra were acquired over m/z range from 350 to 1300 for 1 s and one subsequent MS/MS spectra from 80 to 1800 m/z for 1.5 s. Selected precursor ions were excluded for 50 s from the analysis.

Protein Identification—
MS/MS data were extracted using the AnalystQS software, version 1.0SP8, and the vendor provided script Mascot.dll, version 1.6b16 (Applied Biosystems). The header information of the resulting peak list was modified based on the requirements of MsQuant, version 1.4a16 (29), using an in-house Practical Extraction and Report Language (PERL) script. Peptides were identified by searching the peak-list against the NCBInr database (version 14_08_2006, 486696 mammalian entries) and SwissProt database (version UniProt_Knowledgebase_Release_8.4, 41327 mammalian entries) using the MASCOT algorithm, version 2.103 (Matrix Science, London, UK). The taxonomy parameter was restricted to mammalia, trypsin cleavage specificity was allowed one missed cleavage, peptide tolerance was limited to 50 ppm, fixed modifications were carbamidomethylation of cysteine, variable modifications were oxidation of methionine, and peptides with a score below 18 were excluded. All proteins (except myogenin and myotilin) were identified by MASCOT by at least two peptides in two independent samples with a summed ion score above 45. To estimate the false positive rate, the data set was searched against a reverse database. We observed a false positive rate below 1% using the specified criteria. Because peptide assignments in MASCOT are not always exclusive to a single protein isoform, sequences of MYH, myosin light chain, troponins, {alpha}-actinin, or tropomyosin isoforms were aligned using ClustalW (www.ebi.ac.uk/clustalw). Peptides identified by MASCOT were parsed against the alignment using an HTML browser. Resulting isoform specific peptides were visually verified and arrayed for continuous usage in quantitative analyses. For proteins identified or quantified by a single peptide, fragment spectra are presented using the Arcade software tool2 (supplemental figure). The assignment of proteins to sarcomeric substructures is based on previous studies (1, 3, 30, 31).

Quantitative Analysis—
The MASCOT search result file and the generic mass spectrometry data of each muscle allotype were parsed using MSQuant in a no-label setting. To retrieve quantitative data for peptides that were identified in only a single experiment, the resulting MASCOT search result file was cross-correlated with generic mass spectrometric data from other samples in MSQuant similar to the accurate mass and time tag strategy (25). The quantitation result (peptide ion volumes in thomson·s) for each peptide was visually inspected for equal spacing of mass signals over time, regular chromatographic elution profile, and independence of signals from other peptides (32).

Two dilution series covering protein ratios from 1:1 to 1:16 (66.5–1000 fm) of in-solution digested standard proteins (serum albumin (Bos taurus) and alcohol dehydrogenase (Saccharomyces cerevisae)) were calculated as an average of individual peptide ratios. The observed ratios were based on the averaged result of the analysis of three replicates and compared with the expected ratios (see Fig. 1). Protein ratios for EOM versus EDL were calculated as an average of individual peptide ratios, and the presented ratios were based on three biological replicates. To minimize differences between the samples, ratios were normalized based on the protein ratios of actin, enolase-3-ß, and glyceraldehyde-3-phosphate dehydrogenase. The -fold change was calculated as described previously (33). The COV were calculated as the quotient of the standard deviation and the corresponding average.


Figure 1
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  		FIG. 1. To test the accuracy of the relative label-free protein quantitation, a dilution series from 66.5 to 1000 fm protein digest of serum albumin (B. taurus) (A) or alcohol dehydrogenase (S. cerevisae) (B) was analyzed in triplicate. The experimentally observed ratios were compared with the expected ratios. The error bars denote the standard deviation. The function of the trend line and the coefficient of determination were annotated in the left upper corner.

 
Immunohistochemistry—
Twelve-µm-thick frozen sections of EDL and EOM tissue were prepared and fixed as described previously (5, 16). Incubation with monoclonal anti-mouse antibodies for desmin (Sigma), embryonic MYH (MYH3; clone 2B6) (34), {alpha}-actinin (Sigma), EOM-specific MYH (MYH13; clone 4A6) (6), or polyclonal anti-rabbit antibody {alpha}-B-crystallin (Novocastra, Newcastle upon Tyne, UK) was followed by incubation with appropriate secondary antibodies: goat-anti-mouse-Alexa-Fluor-488, goat-anti-mouse-Alexa-Fluor-546, goat-anti-rabbit-Alexa-Fluor-546, or goat-anti-mouse-IgM-Alexa-Fluor-488 (Molecular Probes, Eugene, OR). Pictures of stained sections were taken using a fluorescence microscope Olympus BX51 equipped with a MagnaFire charge-coupled device camera (Olympus, Center Valley, PA) or a Leica TCS SP2 confocal microscope (Leica Microsystems, Exton, PA). Magnification and exposure levels were not changed during the course of the study.

Western Blot Analysis—
For semi-quantitative validation of the expression levels of selected proteins, Western blot analysis was performed using standard techniques and equipment (Bio-Rad). Crude homogenates were prepared, and equal amounts (25 µg or 40 µg) were subjected to one-dimensional gel electrophoresis on pre-cast gradient gels 4–15% for 90 min at 120 V. The proteins were transferred onto a PVDF membrane (1.5 h, 100 V) and probed with the following antibodies: glyceraldehyde-3-phosphate dehydrogenase monoclonal mouse antibody (Chemicon, Temecula, CA), {alpha}-B-crystallin monoclonal mouse antibody (GeneTex, San Antonio, TX), MYH3 embryonic MYH monoclonal mouse antibody (MYH3; clone 2B6) (34), or MYH7 monoclonal mouse antibody (Myh7; clone NOQ7.5.4D). Secondary antibodies (anti-mouse antibodies) conjugated with horseradish peroxidase were used in combination with enhanced chemiluminescence and detected on autoradiography films (GE Healthcare, Pittsburgh, PA).


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Label-free Protein Quantitation—
To benchmark the accuracy of the protein quantitation strategy and instrumentation, a dilution series of two different proteins was analyzed. Two different peptides for serum albumin and three different peptides for alcohol dehydrogenase were quantified. The expected ratio was plotted against the observed ratio, and a trend line was fitted.

The angle of the trend line deviated –3.1° for serum albumin and –0.7° for alcohol dehydrogenase from the ideal line of 45°. The decreased slope of the trend lines indicated that the experimental setup would underestimate a -fold change. This is especially so for higher values mainly because of saturation of the ion detection system.

The error of the observed ratios to the expected ratios was on average 6.2 and 6.3%, respectively. This is comparable with the performance of previous studies (24, 35, 36) applying different quantitation strategies and instrumentation.

Assignment of Isoform Specific Peptides—
We studied in more detail the assignment of peptides to the various MYH by MASCOT, and here we show how an array of isoform specific peptides can improve the analysis. The MYH are particular problematic because the protein family is highly abundant, generates a large number of tryptic peptides, and the eight isoforms present in striated muscle have sequence identities ranging from 95% for MYH1 and MYH2 to 75% for MYH7 and MYH13.

Fig. 2 shows the number of peptides identified by MASCOT and the number of isoform specific peptides present in the array for a data set from EOM and EDL. The number of overlapping peptides assigned to an isoform decreases as the isoform is reported at a later position in the MASCOT result file. The exception to this trend is MYH7. Because previous studies have suggested that MyCH6 is present in rat EOM (12), its sequence was included in the multiple sequence alignment of MYH. Several peptides reported by MASCOT for MYH7 overlapped with the peptides from MYH6 and therefore were excluded. Even though MASCOT could not clearly determine isoform specific peptides, MASCOT was able to show that MYH8 and MYH13 are present only in EOM (Fig. 2).


Figure 2
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  		FIG. 2. The different myosin heavy chain isoforms are sorted in the order they have been reported as protein identification hits in a representative MASCOT search result file for an EOM or EDL sample. The total height of each bar corresponds to the number of unique peptides reported by MASCOT as "bold red." The black portion of each bar represents the number of isoform specific peptides. MASCOT was able to identify MYH8 and MYH13 only in EOM. This result correlates well with previous studies (12). For EOM, the percentage of isoform specific peptides increases from 21% for MYH4 to 100% for MYH3. It drops again to 60% for MYH7 because of its high sequence identity to MYH6 (not detected in rat EOM or EDL). For EDL, the same trend was observed.

 
For MYH4, the use of isoform specific peptides (10 peptides) instead of peptides identified by MASCOT (48 peptides) decreased the COV of the intensity ratios of all peptides from 73.8 to 16.8%. This approach was further used for identification of isoform specific peptides from myosin light chain, tropomyosin, troponin T, and {alpha}-actinin with similar results.

Data Normalization—
The EOM and EDL of three biological replicates were analyzed. In total, 46 proteins were quantitated and presented in Table II. Forty proteins had been previously associated with the sarcomere (1, 30, 31). Only one sarcomeric protein, PDZ and LIM domain protein 5, was identified but could not be quantitated. Before data normalization the average COV was 19.6%, nine proteins binned into ratios from –1.5 to +1.5, and only three into the bin from ±1.2. To improve this situation, we used actin, enolase-3-ß, and glyceraldehyde-3-phosphate dehydrogenase as internal standards and normalized each sample based on these three ratios. This improved the COV to 17.3%, resulting in 27 proteins with ratios from ± 1.5 and 20 proteins with ratios from ±1.2. Fig. 3 shows the distribution of -fold changes after data normalization. Based on data distribution, COV, the dynamic range of the MS, and previous experience to biological relevance of quantitation results, the significance threshold was set to an abundance ratio of ±1.5. Seventeen sarcomeric proteins showed a -fold change above the threshold level.


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  		TABLE II Proteomics profile of extraocular muscle versus extensor digitorum longus

The ratio (proteomics) and the coefficient of variation are based on the analysis of EOM versus EDL from three different Sprague-Dawley rats. The number of peptides used for quantitation and the molecular mass (MM) are annotated in the third and fourth columns, respectively. Proteins that are differentially regulated are presented in bold. In the case of MYH8 and MYH13, no signal could be detected in EDL samples. Therefore ratios of >25 were annotated. This corresponds to the upper limit of the dynamic range of the experimental setup. In total 46 proteins have been quantitated; 19 of them showed significant -fold changes. Accession numbers are from the NCBInr or SwissProt database.

 

Figure 3
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  		FIG. 3. Normalized -fold changes for the 44 proteins profiled in this study. MYH8 and MYH13 have been excluded. Abundance ratios for each protein were normalized as described above. The shown -fold change was calculated by subtracting a factor of one from the absolute abundance ratio value. Consequently, a -fold change of 0.5 is to be considered significant. Error bars for each protein indicate the standard deviation for the abundance ratios from three biological replicates.

 
Correlation with Microarray Data—
Two groups conducted a total of four microarray experiments in the last few years (5, 8, 9, 11). In these studies different species and limb muscles were assessed. Transcriptomics discovered 10 proteins to be differentially regulated in EOM versus limb muscle (Table I). Five of these proteins overlapped with proteins showing -fold changes from our proteomics study. These include key differences of specific MYH isoforms (MYH3, MYH8, and MYH13) and a regulatory muscle protein (SLIM). In five cases, microarray reported a -fold change where proteomics analysis showed equal amounts in both allotypes.


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  		TABLE I Correlation with microarray data

The averaged ratio (microarray) of EOM versus EDL is calculated along the coefficient of variation based on ratios from previous microarray studies. Significant abundance ratios are marked in bold letters. The superscripted index indicates the origin of the ratios used for the calculation. The limb muscle and species analyzed are annotated next to the original publication. Five proteins show significant changes in proteomics and microarray analysis. >25 corresponds to the upper limit of the linear range of the proteomics setup.

 
Proteomics Profiling of the Sarcomere—
Of the 40 proteins associated with the sarcomere, 24 did not show a significant difference (Table II). These proteins include the troponins, most myosin light chains, {alpha}-actin, and tropomyosin isoforms. This indicates that the basic molecular machinery necessary for muscle contraction is not significantly altered in EOM. In contrast, proteins connecting the Z-band to the thin filament and the costameres are up-regulated in EOM. These proteins include vimentin, desmin, plectin, {alpha}-B-crystallin, and {alpha}-actinin.

In rat EOM, seven MYH were identified ranging from slow ß-cardiac MYH (MYH7) to super-fast EOM-specific MYH (MYH13). Previous one-dimensional gel electrophoresis approaches failed to resolve all MYH present in EOM because of their close mass range (13, 14), and most data concerning protein abundance were gained by immunohistochemistry (12).

Although EOM-specific MYH (MYH13) and perinatal MYH (MYH8) were exclusive to EOM, trace amounts of embryonic MYH (MYH3) could be found in rat EDL. In both muscle allotypes MYH IIx/d (MYH1), IIa (MYH2), IIb (MYH4), and ß-cardiac MYH (MYH7) were present. Of these proteins only MYH1 showed a significant reduction in EOM. In the multiple innervated muscle fibers of EOM a slow tonic MYH was suspected that allows for tonic fiber contraction. The identification of this isoform proved to be difficult because of cross-reactivity of ß-cardiac MYH antibodies and the lack of sequence information.3 Without sequence information mass spectrometry did not fare better in this task.

Immunohistochemistry—
Immunohistochemistry was performed for MYH13, MYH3, {alpha}-actinin, desmin and {alpha}-B-crystallin on sections of EOM and EDL. The left column shows sections of EOM (Fig. 4, A–E'). In transverse sections, the outer orbital layer is oriented to the right and characterized by a smaller fiber diameter (Fig. 4, A–E). The right column shows EDL sections (Fig. 4, F–K').


Figure 4
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  		FIG. 4. The left column shows EOM sections (A–E'); the right column shows EDL sections (F–K'). MYH13 antibodies label predominantly myofibers in orbital layer of EOM (A) and do not label EDL (F). Anti-MYH3 antibody labels exclusively myofibers in the orbital layer (B) and does not label EDL fibers (G). {alpha}-Actinin antibodies stain EOM (C) brighter than EDL (H). Anti-desmin shows more intense staining in EOM (D) than EDL (I), where some fibers are positive, some are weakly stained or negative (D and I). Confocal micrographs show desmin staining in a regularly alternating periodicity following sarcomeric structure. Staining is slightly weaker and less structured in EDL (I') compared to the EOM (D'). {alpha}-B crystallin antibodies strongly label myofibers in EOM, especially in the orbital layer (E). Labeling of EDL myofibers is less intense, and only some fibers are positive (K). Confocal micrographs show {alpha}-B-crystallin staining pattern resembling sarcomeric structure (I'). The structure seems to be less intense and ordered in EDL (K'). Scale bar (fluorescence microscopy) 100 µm; scale bar (confocal microscopy) 10 µm (D', I', E', and K').

 
Antibody against EOM-specific MYH (MYH13) stained predominantly the orbital layer, whereas MYH3 specific antibody stains exclusively the orbital layer as described previously (12). Anti-{alpha}-actinin antibody stained EOM stronger than EDL (Fig. 4, C and H) with a preference for the orbital layer of EOM. {alpha}-Actinin did not seem to vary considerably in neighboring fibers in both muscle allotypes. For desmin and {alpha}-B-crystallin, EOM shows more intense staining than EDL (Fig. 4, D, E, I, and K).

Desmin and in particular {alpha}-B-crystallin seem to be enriched in the orbital layer (Fig. 4, D and E). In both muscle allotypes staining intensity varied strongly in adjacent fibers, which might be caused by the unique pattern of striated muscle. Confocal microscopy of longitudinal sections was performed for both proteins to address this issue. Both antibodies revealed the muscle specific striation pattern in the two allotypes (Fig. 4, D', I', E', and K'). Staining was more intense in EOM than in EDL for desmin and especially {alpha}-B-crystallin. Additionally, the striation pattern seems to be more ordered, and individual bands seem to be closer to each other in EOM. The differences in immunohistochemical staining intensities in EOM versus EDL were in good correlation with the results of our proteomics study.

Western Blot Analysis—
To further validate the results of the proteomics profiling, semi-quantitative Western blot analysis of selected proteins was performed. The results presented in Fig. 5 correlate well with the data presented for the proteomics profile of sarcomere associated proteins (Table I). MYH3 showed strong over-expression for EOM in both Western blot and mass spectrometry-based analysis. For MYH7, both techniques showed only a slight and insignificant increase in EOM. {alpha}-B-crystallin showed elevated levels in both analyses, whereas glyceraldehyde-3-phosphate dehydrogenase levels did not vary significantly in either muscle allotypes.


Figure 5
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  		FIG. 5. Western blot analysis was performed on equal amounts of tissue homogenate to further validate the results of the proteomics profiling. The left column represents EOM, and the right column represents EDL. The different lanes show A, MYH3; B, MYH7; C, {alpha}-B-crystallin; and D, glyceraldehyde-3-phosphate dehydrogenase. The ratio determined by proteomics of EOM versus EDL is presented to the right. MYH3 and {alpha}-B-crystallin show strong up-regulation in EOM. MYH7 and glyceraldehyde-3-phosphate dehydrogenase display only a minor expression difference in both muscle allotypes. The presented expression differences correlate with the results of the label-free protein quantitation.

 

   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The database search algorithm MASCOT assigns sequenced peptides by the principal of parsimony. This approach attempts to find the minimal set of proteins accounting for most sequenced peptides. Simplified, the protein to which most peptides can be assigned will be reported as the highest scoring protein identification. In the case of closely related protein isoforms, peptides generated by all or several isoforms will be assigned to the highest scoring protein isoform they match. Lower scoring isoforms will have a lower number of overlapping peptides assigned to them. Although this allows one to differentially identify closely related protein isoforms, it leads to problems if peptides are used for protein quantitation (Fig. 2). Because characteristics of sarcomeres and subsequent muscle fibers are defined by closely related protein isoforms, this is especially relevant for the quantitation of sarcomeric proteins. Although it is less time consuming in the case of proteins identified by only one or two peptides to use a Basic Local Alignment Search Tool (BLAST) (37) search to validate the isoform specificity, an array based on a multiple sequence alignment saves considerable time while improving the quantitation result when several dozen peptides have to be considered in a larger number of related experiments.

Over the last few years, microarray technology has generated most data on expression differences in a variety of biological systems. The advantage of proteomics measurements is that for most systems functional and structural events occur at the protein level, and changes on the mRNA level do not necessarily translate quantitatively. -Fold changes reported by microarray analysis overlapped in five cases with proteins showing -fold changes from our proteomics study (Table I). These include key differences concerning the presence of specific MYH isoforms (MYH3, MYH8, and MYH13) and a regulatory muscle protein (SLIM). SLIM was shown to decrease with muscle contractile speed (2). Even though EOM contains a large number of slow muscle fibers, it is still considered one of the fastest muscles in the vertebrate body (15). One-dimensional electrophoresis previously showed that in EDL, a fast isoform (MYH4) accounted for ~45% of all MYH, whereas in EOM fast MYH (MYH4 and MYH13) accounts for 75% (13, 18, 38). Considering the higher amount of fast MYH in EOM, the elevated expression level of SLIM in EOM correlates well. SLIM still has a number of controversial molecular functions. The suggested role of SLIM ranges from stabilizing fiber integrity with its binding partner {alpha}-actinin (39), regulating muscle development by interacting with MyoD (40), to being relevant in stretch proprioception (41).

In five cases, microarrays reported a -fold change where proteomics analysis showed equal amounts in both allotypes. Four of these -fold changes were only reported in a single microarray study or showed an extremely high COV. A possible explanation is that these -fold changes are either false positives or species/tissue specific gene products. In the case of slow ß-cardiac MYH (MYH7), microarray studies compared slower limb muscles (soleus, gastrocnemieus, and tibialis anterior) to EOM. Therefore it is not surprising that these studies found increased mRNA level of MYH7 in those skeletal muscles, although we found comparable amounts of MYH7 in both EOM and EDL. This is supported by studies using one-dimensional electrophoresis showing equal amounts of MYH7 in EOM and EDL (13, 18, 38). Additionally, we showed in a previous study using semi-quantitative reverse-transcription polymerase chain reaction muscle development regulatory protein myogenin is up-regulated in some muscle fibers of EOM (27). Thus, our proteomics results are concordant with previous studies.

Based on serial analysis of gene expression (SAGE) data, Porter and colleagues (11, 15) suggested that several key structural proteins (desmin, titin, vimentin, and nebulin) are of low abundance or even absent in EOM. This is not supported by the data in this study (Table II). Intriguingly, we observed the opposite. In EOM, proteins connecting the Z-band to the thin filament and the costameres are augmented. These proteins include vimentin, desmin, plectin, {alpha}-B-crystallin, and {alpha}-actinin. Even though all these proteins are not essential for myofiber development during embryogenesis, they are all necessary for correct fiber organization and stabilization as soon as contraction begins. {alpha}-Actinin cross-links both individual thin filaments and peripheral thin filaments to titin from neighboring sarcomeres (31). A recent study (42) showed that {alpha}-actinin plays a central role in the interaction of major elements of the muscle cell at the Z-band, and based on their experimental results an increased pool of {alpha}-actinin might be important for transmission of forces, better fiber maintenance, formation of stress fibers, and assembly of myofibrils. Transverse sections showed increased amounts of {alpha}-actinin in EOM and EDL (Fig. 4). In both muscle allotypes, little variation in staining intensity in neighboring fibers was observed. The dynamic exchange between the {alpha}-actinin pool and the Z-band described by Sanger and colleagues (42) in quail myotubes could account for this.

The Z-band itself is connected to the costameres in the sarcolemma by desmin, {alpha}-B-crystallin, and plectin. All of these proteins show elevated protein levels when compared with limb muscle. Most muscles in desmin–/– knock-out mice are remarkably unaffected, whereas highly active muscles developed muscle dystrophy by disintegration of the Z-band and loss of membrane integrity (43). Human patients affected by desmin-related myopathy suffer from slowly progressive skeletal muscle weakness, cardiomyopathy, and abnormal ectopic accumulation of desmin-associated proteins (44). Desmin-related myopathies are also caused by mutations in the small chaperone {alpha}-B-crystallin, and a similar phenotype is associated with alterations in the plectin gene (45, 46). What implication does the stoichiometric increase of the desmin/{alpha}-B-crystallin/plectin-complex have for EOM physiology? This can be best answered by extrapolating data from a thorough study of the effects of transgenic loss of desmin on the functionality of mouse diaphragm (43). The diaphragm does not generate super fast movements like EOM, but it has a similar high activity under a higher load. Considering once more the high demand of ocular movements, special mechanisms are necessary to maintain the structural integrity in EOM. The muscle fibers are subject to repetitive cycles of rapid contraction and lengthening (or eccentric contractions) during saccadic eye movements. The unique distribution of MYH and other contractile proteins leads to considerable differences of maximum twitch force production in EOM, in turn leading to biaxial loading (12, 43). Desmin is able to dissipate this energy by contributing to muscle viscoelasticity and modulation of twitch stress production. Further, desmin couples longitudinal and transverse structural elements making the muscle more extensible in the transverse plane, thus preventing myofiber tearing under transverse and biaxial loading. Compared with limb muscle, diaphragm and EOM show elevated protein levels of desmin (43), an adaptation to the special requirements of these two muscle allotypes concerning force distribution and endurance. Further, desmin's embryonic ancestor, vimentin, is also present in increased amounts in EOM. In our study, the molecular chaperone {alpha}-B-crystallin showed the highest -fold change of all Z-band associated protein. {alpha}-B-crystallin has been shown to be necessary to help desmin and vimentin form functional intermediate filaments and prevent pathological aggregation of desmin and vimentin (47). Therefore we suggest that the increase of Z-band associated proteins helps EOM fiber to retain its structural integrity under the high forces occurring during ocular movements. The physiology of these changes will be addressed in future studies.

Approximately 65 proteins have been associated with the sarcomere over the last years (1, 3, 30, 31). In this study only 40 sarcomeric proteins could be identified. Why is this the case? Isoforms like the {alpha} cardiac MYH (MYH6) and cardiac specific myosin-binding protein C might just not be present in EOM and EDL. Cardiac and fast troponin T isoform could not be differentiated by their peptides because of high sequence identity. In the case of core structural or functional proteins like obscurin or the muscle specific ring finger proteins the problem is more of a technical nature. The instrumentation used here is still very able to identify peptides generated by highly abundant proteins like the MYH or troponins but has issues with lower abundant ones. MYH isoforms could still be identified by several isoform specific peptides from amounts corresponding to 25 µg of lysate, making single fiber analysis feasible. In the case of myogenin or myotilin only a single peptide could be sequenced because of technical limitations of the instrumentation. Recent developments in mass spectrometry allow for higher sensitivity, larger dynamic range and more sequencing events per second than the instrumentation in this study (48, 49). Taking these technological developments into account, the number of quantitated sarcomeric proteins could be above 50 in combination with better sequence coverage.

The presented approach is already an improvement on current technologies studying the quantitative composition of the sarcomere. The approach allows the analysis of a large part of the proteome of the sarcomere using a simple protein extraction protocol and a fast analysis by a nowadays widely available mass spectrometry instrument in combination with open source software. This should enable future studies to assess the dynamics, physiology, and pathology of the sarcomere in different muscle allotypes in greater detail.


   ACKNOWLEDGMENTS
 
We thank Konrad Förstner for assistance with the Practical Extraction and Report Language (PERL). We thank Anushka Bangara for proofreading the manuscript.


  FOOTNOTES
 
Received, September 5, 2006, and in revised form, December 15, 2006.

Published, MCP Papers in Press, January 16, 2007, DOI 10.1074/mcp.M600345-MCP200

1 The abbreviations used are: EOM, extraocular muscle; MYH, myosin heavy chain; EDL, extensor digitorum longus; COV, coefficient of variation; SLIM, skeletal muscle LIM protein; fm, fmol. Back

2 M. Wilm, unpublished data. Back

3 N. A. Rubinstein, unpublished data. Back

* This work was partially supported by grants from the National Institutes of Health (AR48871 and EY13862 to T S K. and EY11779 to N A R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

S The on-line version of this article (available at http://www.mcponline.org) contains supplemental material. Back

|| Supported by an internal fellowship from the European Molecular Biology Laboratory. To whom correspondence should be addressed: Gene Expression Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany. E-mail: fraterma{at}embl.de; Tel.: 49-6221-387-8224; Fax: 49-6221-387-306


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