Single-step inline hydroxyapatite enrichment facilitates identification and quantitation of phosphopeptides from mass-limited proteomes with MudPIT

J Proteome Res. 2012 May 4;11(5):2697-709. doi: 10.1021/pr300200x. Epub 2012 Apr 17.


Herein we report the characterization and optimization of single-step inline enrichment of phosphopeptides directly from small amounts of whole cell and tissue lysates (100-500 μg) using a hydroxyapatite (HAP) microcolumn and Multidimensional Protein Identification Technology (MudPIT). In comparison to a triplicate HILIC-IMAC phosphopeptide enrichment study, ∼80% of the phosphopeptides identified using HAP-MudPIT were unique. Similarly, analysis of the consensus phosphorylation motifs between the two enrichment methods illustrates the complementarity of calcium- and iron-based enrichment methods and the higher sensitivity and selectivity of HAP-MudPIT for acidic motifs. We demonstrate how the identification of more multiply phosphorylated peptides from HAP-MudPIT can be used to quantify phosphorylation cooperativity. Through optimization of HAP-MudPIT on a whole cell lysate we routinely achieved identification and quantification of ca. 1000 phosphopeptides from a ∼1 h enrichment and 12 h MudPIT analysis on small quantities of material. Finally, we applied this optimized method to identify phosphorylation sites from a mass-limited mouse brain region, the amygdala (200-500 μg), identifying up to 4000 phosphopeptides per run.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acids / chemistry
  • Amino Acid Motifs
  • Amino Acid Sequence
  • Amygdala / chemistry
  • Animals
  • Binding Sites
  • Calcium / chemistry
  • Chemical Precipitation
  • Consensus Sequence
  • Durapatite / chemistry*
  • Female
  • Mice
  • Mice, Inbred C57BL
  • Molecular Sequence Annotation
  • Molecular Sequence Data
  • Molecular Weight
  • Nitrogen Isotopes / administration & dosage
  • Nitrogen Isotopes / chemistry
  • Phosphopeptides / chemistry
  • Phosphopeptides / isolation & purification*
  • Phosphorylation
  • Protein Folding
  • Proteome / chemistry
  • Proteome / isolation & purification
  • Proteomics / instrumentation
  • Proteomics / methods*
  • Proteomics / standards
  • Sensitivity and Specificity
  • Solubility


  • Acids
  • Nitrogen Isotopes
  • Phosphopeptides
  • Proteome
  • Durapatite
  • Calcium