Causon, J. | bioRxiv Science

bioRxiv2023

Localization and Quantification of Post-Translational Modifications of Proteins Using Electron Activated Dissociation Fragmentation on a Fast-Acquisition Time-of-Flight Mass Spectrometer

Protein post-translational modifications (PTMs) are crucial and dynamic players in a large variety of cellular processes and signaling, and proteomic technologies have emerged as the method of choice to profile PTMs. However, these analyses remain challenging due to potential low PTM stoichiometry, the presence of multiple PTMs per proteolytic peptide, PTM site localization of isobaric peptides, and labile PTM groups that lead to neutral losses. Collision-induced dissociation (CID) is commonly used for to characterize PTMs, but the application of collision energy can lead to neutral losses and incomplete peptide sequencing for labile PTM groups. In this study, we compared CID to an alternative fragmentation, electron activated dissociation (EAD), operated on a recently introduced fast-acquisition quadrupole-time-of-flight (QqTOF) mass spectrometer. We analyzed a series of synthetic modified peptides, featuring phosphorylated, succinylated, malonylated, and acetylated peptides. We performed targeted, quantitative parallel reaction monitoring (PRM or MRMHR) assays to assess the performances of EAD to characterize, site-localize and quantify peptides with labile modifications. The tunable EAD kinetic energy allowed the preservation of labile modifications and provided better peptide sequence coverage with strong PTM-site localization fragment ions. Zeno trap activation provided significant MS/MS sensitivity gains by an average of 6-11-fold for EAD analyses, regardless of modification type. Evaluation of the quantitative EAD PRM workflows revealed high reproducibility with coefficients of variation of typically [~]2%, as well as very good linearity and quantification accuracy. This novel workflow, combining EAD and Zeno trap, offers confident, accurate, and robust characterization and quantification of PTMs.

biochemistry↗

bioRxiv2022

High-throughput proteomics of nanogram-scale samples with Zeno SWATH DIA

The ability to conduct high-quality proteomic experiments in high throughput has opened new avenues in clinical research, drug discovery, and systems biology. Next to an increase in quantitative precision, recent developments in high-throughput proteomics have also gained proteomic depth, to the extent that earlier gaps between classic and high-throughput experiments have significantly narrowed. Here we introduce and benchmark Zeno SWATH, a data-independent acquisition technique that employs a linear ion trap pulsing (Zeno trap pulsing) in order to increase proteomic depth and dynamic range in proteomic experiments. Combined with the high acquisition speed, these gains in sensitivity are particularly attractive for conducting high-throughput proteomics experiments with high chromatographic flow rates and fast gradients. We demonstrate that when combined with either micro-flow- or analytical-flow-rate chromatography, Zeno SWATH increases protein identification in complex samples 5- to 10-fold when compared to current SWATH acquisition methods on the same instrument. Using 20-min micro-flow chromatography, Zeno SWATH identified > 6,000 proteins from a 62.5 ng load of human cell lysate with more than 5,000 proteins consistently quantified in triplicate injections with a median CV of 6%. Using 5-min analytical-flow-rate chromatography (800 {micro}l/min), Zeno SWATH identified 4,907 proteins from a triplicate injection of 2 {micro}g of a human cell lysate; or more than 3,000 proteins from 250 ng tryptic digest. Zeno SWATH hence facilitates precise proteomic experiments with small sample amounts using a fast and robust high flow-rate chromatographic method, broadening the application space that requires precise proteomic experiments on a large scale.

biochemistry↗