Targeted Quantification of the Lysosomal Proteome in Complex Samples.

Lysosomes are essential organelles involved in cellular homeostasis and metabolic regulation. Prior research has shown that lysosomes contain hundreds of proteins, many of which are present at low abundance. This makes comprehensive proteomic analysis challenging. Established methods have focused on lysosome-enriched fractions to improve detection. However, the ability to quantify lysosomal proteins in complex biological samples remains limited. It was already known that mass spectrometry can identify lysosomal proteins, but the performance of different acquisition strategies in complex samples had not been fully explored. That uncertainty drove the need for a comparative study of DIA and PRM. No prior work had resolved how these methods perform across varying sample complexity and gradient lengths. This gap motivated the current investigation.

The study aimed to compare the effectiveness of DIA and PRM in quantifying lysosomal proteins in complex biological samples. The specific problem addressed was the limited ability to detect low-abundance lysosomal proteins in whole-cell or tissue lysates. The motivation stemmed from the need to understand which acquisition method performs better under different experimental conditions. Researchers proposed to use lysosome-enriched fractions and whole-cell or tissue lysates to evaluate DIA and PRM. The goal was to determine whether one method consistently outperforms the other in terms of protein identification and quantification. The study also aimed to assess how sample complexity and chromatographic gradient length affect performance. This approach allows for a direct comparison of DIA and PRM in real-world biological contexts.

The study used two mass spectrometry acquisition methods: DIA and PRM. Lysosome-enriched fractions were prepared from mouse embryonic fibroblasts. Whole-cell lysates and whole-tissue lysates from mouse liver were also analyzed. Each sample was subjected to either DIA or PRM. Chromatographic gradients of varying lengths were used to assess performance differences. Data were collected using mass spectrometers configured for each method. The data were then analyzed for protein identification and quantification accuracy. Correlation between DIA and PRM results was evaluated to determine consistency across methods.

DIA identified more lysosomal proteins on average compared to PRM, particularly in less complex samples and with longer chromatographic gradients. PRM outperformed DIA in highly complex tissue samples and when shorter gradients were used. Both methods successfully identified and quantified lysosomal proteins across all sample types. The data from DIA and PRM showed strong correlation, suggesting consistency in their findings. DIA's advantage was most pronounced in lower-complexity samples and longer gradients. PRM provided better performance in mouse liver lysates with shorter gradients. The number of identified proteins varied depending on sample type and gradient length. These results suggest that the choice of method should consider sample complexity and gradient duration.

The study found that DIA and PRM each have strengths depending on sample complexity and gradient length. DIA performed better in less complex samples and longer gradients, while PRM was superior in highly complex samples and shorter gradients. Both methods provided reliable quantification of lysosomal proteins. The data correlation between DIA and PRM indicates that results from either method can be trusted. The findings suggest that researchers should choose between DIA and PRM based on their specific experimental conditions. The lysosome-enriched fraction provided a baseline for comparison across methods. The study did not propose new lysosomal functions but clarified the performance of proteomic methods. These results support the use of targeted approaches for lysosomal proteome analysis.