Ion mobility-resolved phosphoproteomics with dia-PASEF and short gradients

Abstract

Mass spectrometry-based phosphoproteomics has identified >150,000 post-translational phosphorylation sites in the human proteome. To disentangle their functional relevance, complex experimental designs that require increased throughput are now coming into focus. Here, we apply dia-PASEF on a trapped ion mobility (TIMS) mass spectrometer to analyze the phosphoproteome of a human cancer cell line in short liquid chromatography gradients. At low sample amounts equivalent to ~20 μg protein digest per analysis, we quantified over 12,000 phosphopeptides including ~8,000 class I phosphosites in one hour without a spectral library. Decreasing the gradient time to 15 min yielded virtually identical coverage of the phosphoproteome, and with 7 min gradients we still quantified about 80% of the class I sites with a median coefficient of variation <10% in quadruplicates. We attribute this in part to the increased peak capacity, which effectively compensates for the higher peptide density per time unit in shorter gradients. Our data shows a five-fold reduction in the number of co-isolated peptides with TIMS. In the most extreme case, these were positional isomers of nearby phosphosites that remained unresolved with fast chromatography. In summary, we demonstrate how key features of dia-PASEF translate to phosphoproteomics, resulting in high throughput and sensitivity.

Peptide collision cross sections of 22 post-translational modifications

“…On a global level, we observed major shifts in the m/z vs. ion mobility distribution for modified peptides, which we attributed to changes in their predominant charge state. In proteomics practice, such effects can be important, for example, to optimize the precursor selection scheme in dia-PASEF experiments (21, 22) or to bias data-dependent acquisition towards modified peptides (24, 44).…”

“…The advantages of IMS in terms of speed, sensitivity and specificity should equally apply to or even be enhanced in the analysis of PTMs, given the additional complexity arising from isobaric modifications and positional isomers (21)(22)(23)(24). This motivated researchers since the beginnings of IMS to study the effect of specific modifications on the gas phase characteristics of model peptides.…”

Mass spectrometry‐based high‐throughput proteomics and its role in biomedical studies and systems biology

“…The dia-PASEF technology likewise shows performance with fast methods, quantifying over 6000 proteins in 11 min [99] and more than 5000 proteins in 4.8 min [100]. Further, DIA-PASEF has been applied to quantify 12,000 phosphopeptides in 15 min measurements [101].…”

“…The first DIA software specifically developed to support fast proteomic experiments, DIA‐NN, uses neural networks to filter out false precursor‐spectrum matches and thus enable confident identification and quantification of peptides and proteins in short gradients [95]. Further, the commercial software Spectronaut [87] has been successfully used for the analysis of short gradient DIA runs [81, 99, 101], demonstrating its applicability for large‐scale and HT discovery studies.…”

“…Further, the commercial software Spectronaut [87] has been successfully used for the analysis of short gradient DIA runs [81,99,101], demonstrating its applicability for large-scale and HT discovery studies.…”