Preparation of Human Skeletal Muscle Samples for Proteomic Analysis with Isobaric iTRAQ Labels
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In the past decade, mass spectrometry studies of skeletal muscles have become common. In this tissue, the abundance of several contractile proteins significantly limits the depth of the panoramic proteome analysis. The use of isobaric labels allows improving assessment of the changes in the protein content, while analyzing up to 10 samples in a single run. Here we present the results of a comparative study of various methods for the fractionation of skeletal muscle peptides labeled with an isobaric label iTRAQ. Samples from m. vastus lateralis of eight young males were collected with a needle biopsy. After digestion into peptides and labeling, the preparations were carried out according to three different protocols: (1) peptide purification, HPLC-MS/MS; (2) peptide purification, isoelectric focusing, HPLC-MS/MS; (3) high pH reverse-phase LC fractionation, HPLC-MS/MS. Fractionation of labeled peptides by high pH reverse-phase LC was the optimal strategy for increasing the depth of the proteome analysis. This approach, in addition to contractile and mitochondrial proteins, allowed us to detect a variety of regulatory molecules, including the nucleic acids binding the proteins, chaperones, receptors, and transcription factors.
Keywords:mass spectrometry proteome skeletal muscle peptide fractionation isobaric label Itraq
This study was supported by the Russian Foundation for Basic Research, project no. 17-00-00308 (17-00-00242).
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Conflict of interest. The authors declare that they have no conflict of interest.
Statement of the welfare of animals and humans as subjects. This study was approved by the Biomedical Ethics Committee of the Institute of Biomedical Problems, Russian Academy of Sciences, Moscow. All participants signed their informed consent for participation in this study.
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