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

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 labelfree, 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. Molecular & Cellular Proteomics 6:728 -737, 2007.

On Marathons and Sprints: An Integrated Quantitative Proteomics and Transcriptomics Analysis of Differences Between Slow and Fast Muscle Fibers

“…The corresponding ratios of both proteins are listed in (Table IA, B). The identification of specific isoforms of myosin heavy chains (MyHC) by mass spectrometry presents a more challenging problem because extensive sequence homologies exist between different MyHC proteins (32). We took a conservative approach and used only unique peptides for protein quantification to avoid any misinterpretation of our data set (Fig.…”

Translation initiation by the c-myc mRNA internal ribosome entry sequence and the poly(A) tail

“…To this end, we combined GRNA chromatography (Czaplinski et al 2005;Duncan et al 2006) with quantitative label-free shotgun mass spectrometry (Fraterman et al 2007). Interestingly, we specifically identify eIF4GI but not eIF4GII within c-myc IRES-containing 48S complexes (Fig.…”

“…All peptides were identified with a MASCOT score above 18. Peptide sequences were checked for isoform specificity as previously described (Fraterman et al 2007). For each protein the number of fragment spectra matched by MASCOT is shown in Table 1.…”

“…4A; Czaplinski et al 2005;Duncan et al 2006). The eluted complexes were analyzed by quantitative label-free shotgun proteomics (Fraterman et al 2007). Importantly, this strategy allows validation of the 48S ribosomal complexes by determining the enrichment of small versus large ribosomal subunit proteins.…”

“…Peptides were trypsin-digested in solution and separated on a nano-flow 1D-plus Eksigent HPLC system coupled with a QStar Pulsar i quadrupole Time-of-Flight MS (Applied Biosystemsy) as described (Fraterman et al 2007). Peptides were identified by searching the peak-list against the IPI human (3.19_20060712, 60,937 sequence entries) database using the MASCOT v2.103 (Matrix Science) algorithm and standard search parameters.…”