Input normalization and PTM stoichiometry calculations in Spectronaut^®

Introduction

Post-translational modifications (PTM) such as protein phosphorylation can dramatically alter the function of proteins and have important roles in disease and health as biomarkers or pharmaceutical targets. An important aspect of PTMs is the rate of site occupancy or PTM stoichiometry, which can provide biological information irrespective of the underlying protein abundance. In proteomics experiments where modifications are studied using an enrichment step, it is important to decouple the changes in PTM site quantity from the changes in protein abundance in the original sample. This is achieved by performing input normalization of PTM site quantity. There is currently a need for a software tool that supports input normalization and PTM site occupancy calculations using label-free data-independent acquisition (DIA) measurement of samples which provide simple, deep, and accurate quantification of PTM sites. In this abstract, we present the novel support for input normalization and PTM site occupancy in Spectronaut® for label-free DIA data, in addition to the existing PTM localization support.

Methods

Two DIA phosphoproteome datasets were obtained from a public repository (1). The first dataset used to validate input normalization consists of two mixtures of yeast and human cell extract in 0.25:1 and 2:1 ratios (yeast:human), enriched with Ti-IMAC beads and measured by DIA. The second dataset used to validate PTM stoichiometry consists in enriched yeast phosphopeptides treated with phosphatase or untreated. Phosphopeptides and phosphatase-treated phosphopeptides were mixed in knowns ratios at 0.1%, 1%, 10%, 50%, 90%, 99% and 99.9% stoichiometry with a background of human cell extract. For both datasets, we analyzed the datasets with default settings and phospho (STY) in Spectronaut® 19. Runs were analyzed once to generate reference saved experiments, which were used as input normalization in a second analysis using "All peptides" as input normalization strategy. Stoichiometry was calculated with maximum Q-value of 1% and minimum fold change of 5.

Results

In the first dataset, we measured a 7.32 median fold change between the two samples for yeast PTM site quantities and a 1.03 median fold change after input normalization, effectively showing that the difference in PTM site quantities is due to the difference in protein abundance and not in PTM site stoichiometry. In the second dataset, we achieve 8.8% accuracy and 24.2% precision in the reported PTM stoichiometry compared to the expected PTM site stoichiometries.

Conclusion

The new support for input normalization and PTM site occupancy in Spectronaut® allows scientists and researchers to quickly access relevant and meaningful biological information from their enrichment experiments calculate PTM site occupancy even when protein abundance varies between samples.

References (1) Bekker-Jensen D. B et al., Nature Communications 11:787 (2020)