Optimization of Instrument Parameters for Efficient Phosphopeptide Identification and Localization by Data-dependent Analysis Using Orbitrap Tribrid Mass Spectrometers

10 February 2023, Version 2
This content is a preprint and has not undergone peer review at the time of posting.


The analysis of protein phosphorylation site identification by mass spectrometry-based methods continues to improve with increased efficiency at multiple points of the pipeline including affinity isolation, sample handling with automated protein digestion and phosphopeptide extraction routines, and more sensitive phosphopeptide detection capabilities. The role mass spectrometry parameters play in the quantity and quality of results is understudied, with some reports presenting improvements without sufficient details of how these parameters were derived, nor how they were fully optimized. Here, we systematically scrutinize and optimize parameters of two Orbitrap Tribrid mass spectrometers by varying instrument parameters at both the MS1 and MS2 levels using Titanium IMAC-enriched phosphopeptide samples to illustrate how the recovery of phosphopeptides, and the confidence of phosphosite localizations, vary accordingly. These optimized values are then used to compare different IMAC beads for phosphopeptide-enrichment, and optimal bead combinations used to further improve phosphopeptide detection. These results have implications for planning detailed phosphoprotein identification and quantitation experiments to provide confidence in detection, site localization and reproducibility.


post-translational modification
Method optimization
protein phosphorylation
peptide phosphorylation
phosphosite localization
phosphopeptide enrichment
false discovery rate
false localization rate
mass spectrometry
data-dependent analysis


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