Comprehensive analysis of tropomyosin isoforms in skeletal muscles by top-down proteomics

Abstract

Mammalian skeletal muscles are heterogeneous in nature and are capable of performing various functions. Tropomyosin (Tpm) is a major component of the thin filament in skeletal muscles and plays an important role in controlling muscle contraction and relaxation. Tpm is known to consist of multiple isoforms resulting from different encoding genes and alternative splicing, along with post-translational modifications. However, a systematic characterization of Tpm isoforms in skeletal muscles is still lacking. Therefore, we employed top-down mass spectrometry (MS) to identify and characterize Tpm isoforms present in different skeletal muscles from multiple species, including swine, rat, and human. Our study revealed that Tpm1.1 and Tpm2.2 are the two major Tpm isoforms in swine and rat skeletal muscles, whereas Tpm1.1, Tpm2.2, and Tpm3.12 are present in human skeletal muscles. Tandem MS was utilized to identify the sequences of the major Tpm isoforms. Furthermore, quantitative analysis revealed muscle-type specific differences in the abundance of un-modified and modified Tpm isoforms in rat and human skeletal muscles. This study represents the first systematic investigation of Tpm isoforms in skeletal muscles, which not only demonstrates the capabilities of top-down MS for the comprehensive characterization of skeletal myofilament proteins but also provides the basis for further studies on these Tpm isoforms in muscle-related diseases.

Comprehensive characterization of monoclonal antibody by Fourier transform ion cyclotron resonance mass spectrometry

“…Nevertheless, bottom-up MS may suffer from the introduction of artifacts from lengthy sample preparation, limited protease choice for full protein sequence coverage, and loss of proteoform information in convoluted peptide pools.- 17,18 In contrast to the bottom-up approach, top-down MS analyzes intact proteins directly with minimum sample preparation, which provides an overview of major proteoforms 19 and minimizes sample artifacts. 20 However, the large size of mAbs (~150 kDa) together with intra-and inter-chain disulfide bonds restrict efficient fragmentation of intact proteins, and therefore only~15-35% bond cleavage can be achieved in top-down MS/MS analysis. [21][22][23][24] In addition to bottom-up and top-down approaches, middle-down MS is also commonly used in the characterization of mAbs by analyzing subunits derived from limited protein proteolysis or disulfide bond reduction.…”

Myopathic mutations in the β‐chain of tropomyosin differently affect the structural and functional properties of ββ‐ and αβ‐dimers

“…The isoform Tpm 1.1 (a-Tpm, TPM1 gene product) is expressed in cardiac and skeletal muscles, Tpm 2.2 (b-Tpm, TPM2 gene product), is expressed in fast and slow skeletal muscles, and Tpm 3.1 (g-Tpm, TPM3 gene) is expressed only in slow skeletal muscles (15,16).…”

Identification and Quantification of Proteoforms by Mass Spectrometry

“…This function allows users to perform manual correction of charge states and isotopic masses of fragment ions for comprehensive characterization of sequence variations and PTM sites . The software also offers quantitative tools to evaluate the relative abundances of different proteoforms under various experimental conditions …”