Optimization of label-free nano LC-MS/MS analysis of the placental proteome. (November 2020)

• Record Type: Journal Article
• Authors: Luyten, Leen J.; Dieu, Marc; Demazy, Catherine; Fransolet, Maude; Nawrot, Tim S.; Renard, Patricia; Debacq-Chainiaux, Florence
• Is Part Of: Placenta. Volume 101(2020)
• Page Start: 159
• Page End: 162
• Publication Date: 2020-11
• Subjects: Placenta -- Proteomics -- Label-free nano LC-MS/MS; ACN Acetonitrile -- DTT Dithiothreitol -- FASP Filter-aided sample preparation -- IAA Iodoacetamide -- Nano LC-MS/MS Nano Liquid chromatographic tandem mass spectrometry -- PBS Phosphate buffered saline -- TFA Trifluoroacetic acid -- UA Urea acetate; Placenta -- Periodicals; Reproduction -- Periodicals; Placenta -- Periodicals; Placenta -- Périodiques; Reproduction -- Périodiques; 612.63
• DOI: 10.1016/j.placenta.2020.09.013
• Languages: English
• ISSNs: 0143-4004

Abstract

Abstract: The placenta can be regarded as a mirror of the events to which the fetus is exposed during development. The placental proteome has been studied with several methodologies differing in sample handling, protein extraction, and processing. We optimized a protocol to analyze the placental proteome by means of label-free nano-LC-MS/MS mass spectrometry with regard to sample treatment, protein extraction, and protein digestion, in order to obtain a high protein concentration for identification of a specific protein signature according to the conditions studied. We recommend mechanical tissue disruption, blood removal prior to protein extraction, and FASP-based or in-gel digestion. Highlights: Standardized protocols are essential for placental proteome characterization. Label-free nano LC-MS/MS allows reproducible and accurate analysis of placenta samples. Sample preparation is crucial, as blood removal increases protein identification. Optimized steps include mechanical extraction and FASP-mediated protein digestion. Five-hour digestion with 1/20 w/w trypsin optimizes protein identification quality.