Absolute Quantification of Lysosomal Proteins by Targeted Mass Spectrometry

Abstract

Lysosomes are low abundant, membrane-encapsulated cytoplasmic organelles that play a pivotal role in the cellular clearance of various types of extra- and intracellular material including the degradation of biological macromolecules; thereby having major implications for cellular metabolism. Around 340 proteins are known to reside within, or be temporarily associated with the lysosome, making up the lysosomal proteome. Quantitative proteomics experiments utilizing mass spectrometry (MS) play a crucial role in gaining comprehensive insights into the organelle’s biology and in understanding its involvement and alterations in various diseases. <br/> However, most studies on lysosomes, or such covering lysosomal proteins using MS, have primarily resulted in relative quantitative data, with researchers investigating changes in protein levels between different samples and experimental conditions. Absolute quantities of lysosomal protein remain largely unknown, although such data can be considered as the most desirable/universally applicable outcome of a quantitative experiment. This study utilized the quantification concatemer (QconCAT) strategy to create an absolutely quantified internal standard for 144 lysosomal proteins, covering the majority of the mouse lysosomal core proteome in an MS experiment. Unlike relative quantitative data, the acquired absolute quantitative data allow not only for the calculation of absolute quantities, but also for the comparison of lysosomal protein amounts between sample types and conditions. In addition to the numerous analytical advantages offered by the internal standard applied, absolute quantitative data hold the promise to enhance our understanding of the molecular relationship between different proteins in and across various samples. To further overcome current shortcomings and limitations associated with untargeted MS experiments and lysosome enrichment, limiting the compatibility of certain sample types with MS analyses, targeted MS approaches for the direct study of lysosomal proteins from whole cell and tissue lysates were developed. As a result, a systematic and unbiased quantitative study across various cell/tissue types of varying complexities, independent of any lysosome enrichment/isolation procedures is now possible. <br/> For the investigation of samples with varying input amounts and sample complexity, first a robust and universally applicable sample preparation workflow was established using the MS-compatible surfactant RapiGest. Here, the peptide/protein concentration emerged as a critical parameter in the sample preparation workflow, influencing the precipitation behavior of the surfactant. Further, for the sample preparation of low-concentrated samples a workaround involving a trigger protein for co-precipitation of the surfactant was presented. In an initial, label-free comparative study quantifying lysosomal proteins from various complex sample lysates using data-independent acquisition and parallel reaction monitoring, it became evident that a targeted MS acquisition strategy such as parallel reaction monitoring, surpasses data-independent acquisition-MS in detecting and accurately quantifying subtle changes of low-abundant (lysosomal) proteins within highly complex sample backgrounds such as tissue lysates. For the absolute quantification of 144 lysosomal proteins from any cell/tissue type of mouse origin a targeted MS assay employing multiple reaction monitoring was developed and the stable isotope-labeled, absolutely quantified internal standard was applied. The third study provides absolute quantitative data on 143 lysosomal proteins in one cell line, lysosome-enriched fractions and four primary cell types, enabling the determination of individual lysosomal protein copy numbers. In mouse embryonic fibroblasts, the lysosomal core proteome composition exhibited a dynamic range spanning three orders of magnitude. The spatial distribution of individual proteins and protein groups between lysosomal and non-lysosomal compartment revealed feature-specific localization patterns, with substantial proportions of membrane proteins and partially hydrolytically active proteins localized in non-lysosomal compartments. Across the different cell types investigated, variations in the abundance of distinct lysosomal protein classes and in proteome composition underscored the functional specialization of individual cell types, with primary cells showing elevated levels of hydrolases/luminal proteins. Moreover, unique expression dynamics of functional protein classes as well as selected proteins within these classes highlighted cell type-specific lysosomal adaptations tailored to their specific functions. qPCR analysis of 51 lysosomal proteins demonstrated that this cell type-specific regulation of the proteome is - with only a few exceptions - predominantly driven by transcriptional control.