Mass Spectrometry-Based Proteomic Analysis of Lysosome-Enriched Fractions

Abstract

Lysosomes are the main degradative organelles of most mammalian cells, playing a crucial role in nutrient metabolism and acting as a signaling hub. Due to the low abundance of lysosomes, several enrichment techniques have been developed to facilitate their investigation. Combination of these approaches with mass spectrometry-based proteomics presents a powerful tool for the unbiased characterization of the lysosomal proteome under various conditions. Different enrichment strategies result in lysosome-enriched fractions with unique characteristics, varying, for example, in protein concentration and buffer composition. Therefore, in order to obtain optimal results, the subsequent strategy for proteomics sample preparation has to be adapted. Here, we describe different methods for the processing of lysosome-enriched fractions for mass spectrometry-based proteomic analyses and provide guidance for the selection of the appropriate strategy. We further elaborate on typical parameters for mass spectrometric analysis and data processing.

Keywords: Lysosomes; Mass spectrometry; Proteomics; RapiGest; SP3; Urea.