Bortel, PatriciaPiga, IlariaKoenig, ClaireGerner, ChristopherMartinez-Val, AnaOlsen, Jesper V2024-07-042024-07-042024-05Mol Cell Proteomics. 2024 May;23(5):100754.http://hdl.handle.net/20.500.12105/20077Improving coverage, robustness, and sensitivity is crucial for routine phosphoproteomics analysis by single-shot liquid chromatography-tandem mass spectrometry (LC-MS/MS) from minimal peptide inputs. Here, we systematically optimized key experimental parameters for automated on-bead phosphoproteomics sample preparation with a focus on low-input samples. Assessing the number of identified phosphopeptides, enrichment efficiency, site localization scores, and relative enrichment of multiply-phosphorylated peptides pinpointed critical variables influencing the resulting phosphoproteome. Optimizing glycolic acid concentration in the loading buffer, percentage of ammonium hydroxide in the elution buffer, peptide-to-beads ratio, binding time, sample, and loading buffer volumes allowed us to confidently identify >16,000 phosphopeptides in half-an-hour LC-MS/MS on an Orbitrap Exploris 480 using 30 μg of peptides as starting material. Furthermore, we evaluated how sequential enrichment can boost phosphoproteome coverage and showed that pooling fractions into a single LC-MS/MS analysis increased the depth. We also present an alternative phosphopeptide enrichment strategy based on stepwise addition of beads thereby boosting phosphoproteome coverage by 20%. Finally, we applied our optimized strategy to evaluate phosphoproteome depth with the Orbitrap Astral MS using a cell dilution series and were able to identify >32,000 phosphopeptides from 0.5 million HeLa cells in half-an-hour LC-MS/MS using narrow-window data-independent acquisition (nDIA).engVoRhttp://creativecommons.org/licenses/by/4.0/PhosphopeptidesProteomicsTandem Mass SpectrometryPhosphoproteinsHumansChromatography, LiquidHeLa CellsProteomePhosphorylationAutomationAtribución 4.0 Internacional3854801923510075410.1016/j.mcpro.2024.1007541535-9484Molecular & cellular proteomics : MCPopen access