title: Single-step inline hydroxyapatite enrichment facilitates identification and quantitation of phosphopeptides from mass-limited proteomes with MudPIT.
abstract: 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.
authors.affiliation: Department of Chemical Physiology, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, California 92037, USA.
authors.forename: Bryan R
authors.initials: BR
authors.lastname: Fonslow
authors.forename: Sherry M
authors.initials: SM
authors.lastname: Niessen
authors.forename: Meha
authors.initials: M
authors.lastname: Singh
authors.forename: Catherine C L
authors.initials: CC
authors.lastname: Wong
authors.forename: Tao
authors.initials: T
authors.lastname: Xu
authors.forename: Paulo C
authors.initials: PC
authors.lastname: Carvalho
authors.forename: Jeong
authors.initials: J
authors.lastname: Choi
authors.forename: Sung Kyu
authors.initials: SK
authors.lastname: Park
authors.forename: John R
authors.initials: JR
authors.lastname: Yates
mesh_terms: D000143:Acids / Q000737:chemistry; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000679:Amygdala / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry; D011232:Chemical Precipitation; D016384:Consensus Sequence; D017886:Durapatite / Q000737:chemistry*; D005260:Female; D051379:Mice; D008810:Mice, Inbred C57BL; D058977:Molecular Sequence Annotation; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009587:Nitrogen Isotopes / Q000008:administration & dosage / Q000737:chemistry; D010748:Phosphopeptides / Q000737:chemistry / Q000302:isolation & purification*; D010766:Phosphorylation; D017510:Protein Folding; D020543:Proteome / Q000737:chemistry / Q000302:isolation & purification; D040901:Proteomics / Q000295:instrumentation / Q000379:methods* / Q000592:standards; D012680:Sensitivity and Specificity; D012995:Solubility
chemical_list: D000143:Acids; D009587:Nitrogen Isotopes; D010748:Phosphopeptides; D020543:Proteome; D017886:Durapatite; D002118:Calcium