Peptidomics analysis of lymphoblastoid cell lines.

Abstract

A key challenge in clinics is the identification of sensitive and specific biomarkers for early detection, prognostic evaluation, and surveillance of disease. A biomarker is defined as a biological substance that can be used to specifically detect a disease, measure its progression, or the effect of a treatment. A biomarker should be easily accessible, and ideally sensitivity and specificity must be sufficient to distinguish between false positives, false negatives, and true positives. To be useful for routine clinical evaluation, a biomarker should be detectable in body fluids (e.g., plasma, serum, urine). A biomarker can be a metabolite, a specific post-translational modification, a lipid, a phospholipid, or a protein. Due to technical advances in the analysis of biomolecules by mass spectrometry (MS), investigations of peptide biomarkers have increased. In contrast to genome, the peptidome is dynamic and constantly changing. Elucidating how the peptides complement changes in a cell type in diseases is crucial to understand how these processes occur at a molecular level. Lymphoblastoid cell lines, derived from blood lymphocytes, represent suitable models for biochemical investigations and biomedical applications because of their stability, the ease of amplification, and long-term preservation. Technological improvements of MS and liquid chromatography (LC) during the last 10 years resulted in the development of highly sensitive approaches for proteomic and peptidomic analyses. Here we provide guidelines for the preparation of the lymphoblastoid cell lines, the extraction of the peptides and their purification. We describe a number of technologies which we developed for the peptidomic profiling of lymphoblastoid cell extracts from patients with leukodystrophies, linked to mutations in the genes encoding the eukaryotic initiation factor 2B (eIF2B; eIF2B-related disorders).