EMERGE Episode 18: Systematic optimization of automated enrichment for sensitive phosphoproteomics
Автор: ReSyn Biosciences
Загружено: 2024-04-25
Просмотров: 225
Описание: Improving coverage, robustness, and sensitivity is crucial for routine phosphoproteomics analysis by single-shot liquid chromatography tandem mass spectrometry (LC-MS/MS) from minimal peptide input. This webinar focuses on systematic optimization of key experimental parameters for automated on-bead phosphopeptide sample preparation with a focus on low input samples, pinpointing critical variables influencing the resulting phosphoproteome coverage. Patricia and team further evaluated how sequential enrichment can boost phosphoproteome coverage, and show that pooling fractions into a single LC-MS/MS analysis can increase the depth of coverage. The team developed a new approach to phosphopeptide enrichment based on stepwise addition of beads, which also boosts phosphoproteome coverage. The optimized phosphoproteomics sample processing pipeline was run on one of the newest generation of mass spectrometers, the Orbitrap Astral, increasing the phosphopeptide coverage 2-fold, allowing for deep phosphoproteomics analysis, without the need to scale up the starting cell amounts.
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