HUMAN SKELETAL MUSCLE SAMPLE PREPARATION FOR PROTEOMIC ANALYSIS WITH ISOBARIC iTRAQ LABELS
In the last decade mass spectrometry studies skeletal muscles became common. In this tissue, an abundance of several contractile proteins significantly limits the depth of the panoramic proteome analysis. The use of isobaric labels allows to improve assessment of the changes in 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 8 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 optimal strategy for increasing the depth of proteome analysis. This approach, in addition to contractile and mitochondrial proteins, allowed us to detect a variety of regulatory molecules, including nucleic acids binding proteins, chaperones, receptors and transcription factors.