title: Citric acid-assisted two-step enrichment with TiO2 enhances the separation of multi- and monophosphorylated peptides and increases phosphoprotein profiling.
abstract: Phosphopeptide enrichment is essential for large-scale phosphoprotein profiling. Titanium dioxide (TiO2) is widely used in phosphopeptide enrichment, but it is limited in the isolation of multiphosphorylated peptides due to their strong binding. In this study, we found that citric acid greatly affects the binding of mono- and multiphosphopeptides with TiO2, which can be used for stepwise phosphopeptide separation coupled with mass spectrum (MS) identification. We first loaded approximately 1 mg of peptide mixture of HeLa cell digests onto TiO2 beads in highly concentrated citric acid (1 M). Then the flow-through fraction was diluted to ensure low concentration of citric acid (50 mM) and followed by loading onto another aliquot of TiO2 beads. The two eluted fractions were subjected to nanoLC-MS/MS analysis. We identified 1,500 phosphorylated peptides, of which 69% were multiphosphorylated after the first enrichment. After the second enrichment, 2,167 phosphopeptides, of which 92% were monophosphorylated, were identified. In total, we successfully identified 3,136 unique phosphopeptides containing 3,973 phosphosites utilizing this strategy. Finally, more than 37% of the total phosphopeptides and 2.6-fold more of the multiphosphorylated peptides were identified as compared to the frequently used DHB/TiO2 enrichment strategy. Combining SCX with CATSET, we identified 14,783 phosphopeptides and 15,713 phosphosites, of which 3,678 were unrecorded in PhosphoSitePlus database. This two-step separation procedure for sequentially enriching multi- and monophosphorylated peptides by using citric acid is advantageous in multiphosphorylated peptide separation, as well as for more comprehensive phosphoprotein profiling.
authors.affiliation: State Key Laboratory of Protein and Plant Gene Research, College of Life Sciences, Peking University, Beijing 100871, China.
authors.forename: Xuyang
authors.initials: X
authors.lastname: Zhao
authors.forename: Qingsong
authors.initials: Q
authors.lastname: Wang
authors.forename: Shuxin
authors.initials: S
authors.lastname: Wang
authors.forename: Xiao
authors.initials: X
authors.lastname: Zou
authors.forename: Mingrui
authors.initials: M
authors.lastname: An
authors.forename: Xuefei
authors.initials: X
authors.lastname: Zhang
authors.forename: Jianguo
authors.initials: J
authors.lastname: Ji
mesh_terms: D002853:Chromatography, Liquid / Q000295:instrumentation / Q000379:methods; D019343:Citric Acid / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D010750:Phosphoproteins / Q000302:isolation & purification*; D020543:Proteome / Q000302:isolation & purification*; D053719:Tandem Mass Spectrometry / Q000295:instrumentation / Q000379:methods; D014025:Titanium / Q000737:chemistry*
chemical_list: D010750:Phosphoproteins; D020543:Proteome; C009495:titanium dioxide; D019343:Citric Acid; D014025:Titanium