SureQuant-based absolute quantification of the Lysosomal mouse proteome

The lysosome, which acts in several ways as the cell's metabolic center, is crucial for macromolecule recycling, nutrient sensing, antigen processing, cell death, and metabolic signaling. Dysfunction of lysosomes can lead to severe conditions, including lysosomal storage disorders, neurodegenerative diseases, cancer, and other metabolic syndromes. For studying lysosomal pathology in these diseases and cellular processes, accurate and reproducible quantification of lysosomal proteins is essential. Due to the low abundance of lysosomes, it is challenging to detect and quantify these proteins using whole proteome shotgun proteomics approaches. One way to overcome this is lysosome enrichment, which is typically limited to certain cell lines, hindering studies in other sample types like body fluids or tissues. Furthermore, so-far only relative quantification data are available for lysosomal proteins, and absolute amounts and stoichiometric ratios remain largely unknown. To enable absolute quantification of mouse lysosomal proteins, we developed a targeted strategy using medium/heavy isotope-labeled internal standards which we generated following the Quantification Concatemer (QconCAT) approach. The QconCATs were designed to represent 143 lysosomal proteins, for which three tryptic peptides each were included, organized into 12 QconCAT proteins. Following construct optimization, we synthesized lysoQconCAT genes that incorporated an isobaric standard peptide for normalization purposes and a His-Tag to facilitate affinity purification. We validated the generated QconCAT standards using shotgun proteomics, assessing the quality, sequence coverage, and labeling efficiencies of the isotope-labeled QconCAT peptides. We successfully expressed and purified these QconCAT standard proteins. Using these standards we established a SureQuant assay on an Orbitrap Fusion Lumos. As SureQuant typically uses high levels of heavy-isotope labeled peptides as trigger for PRM-based label free quantification, absolute quantification is not possible as the difference in intensity between these standards and sample peptides does not match the dynamic range of the mass spectrometer. To address this issue, we further added medium isotope-labeled standards for quantification at close to endogenous levels. This 3plex approach, utilizing the heavy-isotope labeled standards as trigger and medium standards for abundance estimation, allows automated absolute quantification with SureQuant.

With this strategy, we determined absolute abundances of lysosomal proteins in mouse cell line. We optimized the spike-in amount of both heavy and medium isotope-labeled standards to maximize the number of covered peptides for absolute quantification, identifying and quantifying 136 verified lysosomal proteins. This method offers a reliable and sensitive approach to study lysosomal proteins, with possible application in the analysis of cell lines and mouse tissues, to advance our understanding of lysosomal function and disease implications.