Journal of Chromatography Open (Nov 2022)
Micro‐pillar array columns (µPAC): An efficient tool for comparing tissue and cultured cells of glioblastoma
Abstract
Glioblastoma is the most common form of brain cancer in adults and is invariably fatal despite extensive treatment with surgery, radio- and chemotherapy. Research on glioblastoma and other cancers is often based on studies of cells in cultures (commercial or patient-derived). However, the impact of cell culturing on the glioblastoma proteome is not yet studied in detail. To investigate this, comprehensive proteome analyses were performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to compare glioblastoma tissue from three patients with cultured cells derived from these tissues.A 200 cm micro-pillar array column (µPAC) was used for separating peptides, applying a 5-hour LC solvent gradient. For sample preparation, we compared filter aided sample preparation (FASP), fractionation with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and fractionation with off-line high-pH reversed-phase LC (48 fractions concatenated in 8 vials) using nano-LC columns.Fractionation provided higher total protein coverages compared to FASP in combination with µPAC. However, FASP + µPAC was arguably more attractive when considering running times and simplicity. FASP in combination with µPAC also provided similar output compared to fractionation in combination with commonly used columns (15 cm, 2.6 µm C18), exemplifying the resolving power of the pillar array format.Using FASP in combination with µPAC, a total of 8469 proteins were quantified across all samples. Among these were proteins specific to either cell culture samples or tissue biopsies. Overall enrichment of transcription factors and other nucleic acid binding factors was observed in the cell culture samples, while proteins related to the vasculature, extracellular matrix, immune response, and proteases were lost upon culturing. Proteins detected in both tissue biopsies and cultured cells from each patient showed a significant correlation of expression levels, indicating that patient-derived cell cultures retain some specific characteristics of the parent tumor.
