Systematic optimization of automated phosphopeptide enrichment for high-sensitivity phosphoproteomics

Abstract

Improving coverage, robustness and sensitivity is crucial for routine phosphoproteomics analysis by single-shot liquid chromatography tandem mass spectrometry (LC-MS/MS) runs from minimal peptide inputs. Here, we systematically optimized key experimental parameters for automated on-beads phosphoproteomics sample preparation with 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 {micro}g 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). Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=159 HEIGHT=200 SRC="FIGDIR/small/568418v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@f455d9org.highwire.dtl.DTLVardef@130537eorg.highwire.dtl.DTLVardef@1b99287org.highwire.dtl.DTLVardef@42e72e_HPS_FORMAT_FIGEXP M_FIG C_FIG