Two-Step Enrichment Facilitates Background Reduction for Proteomic Analysis of Lysosomes

Lysosomes
constitute the main degradative compartment of most mammalian
cells and are involved in various cellular functions. Most of them
are catalyzed by lysosomal proteins, which typically are low abundant,
complicating their analysis by mass spectrometry-based proteomics.
To increase analytical performance and to enable profiling of lysosomal
content, lysosomes are often enriched. Two approaches have gained
popularity in recent years, namely, superparamagnetic iron oxide nanoparticles
(SPIONs) and immunoprecipitation from cells overexpressing a 3xHA-tagged
version of TMEM192 (TMEM-IP). The effect of these approaches on the
lysosomal proteome has not been investigated to date. We addressed
this topic through a combination of both techniques and proteomic
analysis of lysosome-enriched fractions. For SPIONs treatment, we
identified altered cellular iron homeostasis and moderate changes
of the lysosomal proteome. For overexpression of TMEM192, we observed
more pronounced effects in lysosomal protein expression, especially
for lysosomal membrane proteins and those involved in protein trafficking.
Furthermore, we established a combined strategy based on the sequential
enrichment of lysosomes with SPIONs and TMEM-IP. This enabled increased
purity of lysosome-enriched fractions and, through TMEM-IP-based lysosome
enrichment from SPIONs flow-through and eluate fractions, additional
insights into the properties of individual approaches. All data are
available via ProteomeXchange with PXD048696.