Abstract
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.
Highlights
The sarcomere is the major structural and functional unit of striated muscle
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
Summary
All chemicals were purchased from Sigma unless stated otherwise. Graphs were prepared using Microsoft Excel (Microsoft, Redmond, WA) or GraphPad Prism (San Diego, CA). Peptides derived from digested standard proteins were separated by a similar gradient over 30 min. 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. 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]. Protein ratios for EOM versus EDL were calculated as an average of individual peptide ratios, and the presented ratios were based on. 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)
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