biostudies-literatureBortel PEuropean Commission Marie Sklodowska-Curie ActionsOsterreichische ForschungsgemeinschaftUniversity of ViennaH2020Novo Nordisk Foundation Center for Protein ResearchEuropean CommissionNovo Nordisk Fonden100754https://www.ebi.ac.uk/biostudies/studies/S-EPMC11087715biostudies-literatureUnknown23(5)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 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 confiMolecular & cellular proteomics : MCPSystematic Optimization of Automated Phosphopeptide Enrichment for High-Sensitivity Phosphoproteomics.PMC11087715PI Jesper Velgaard OlsenOlsen JVGerner CMartinez-Val APiga IKoenig CBortel PfalseSystematic Optimization of Automated Phosphopeptide Enrichment for High-Sensitivity Phosphoproteomics.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 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 confi2024-01-01T00:00:00Z2024 May2026-06-02T06:09:28.785Z2026-04-15T03:10:08.827ZS-EPMC110877153854801910.1016/j.mcpro.2024.100754