A new maleimide-bound acid-cleavable solid-support reagent for profiling phosphorylation.
A new chemical strategy for phosphopeptide profiling is reported in this study. Phosphorylation represents one of the most important classes of posttranslational modifications of proteins. Here we report a generalized strategy that employs solid-phase capture and mass-encoding steps to selectively enrich phosphopeptides from complex mixtures. This method exploits conversion of phosphates into thiols and reactive compounds to selectively isolate products of phosphorylation. Selective isolation of phosphopeptides is achieved with a simple, novel, acid-cleavable, solid-support-bound maleimide reagent. Our chemistry efforts have focused on minimization of linker size and simplification of reagent production with incorporation of common solid-phase peptide synthesis steps. Relative quantitation was demonstrated by modifying phosphopeptides with incorporation of ethanedithiol and propanedithiol. We observed that appropriate normalization is necessary to utilize mass tag strategies for relative quantitation of posttranslational modifications. The utility of solid-phase capture was determined with model phosphopeptides, and the method was demonstrated with enriching phosphopeptides from beta-casein, alpha-casein and ovalbumin. The solid-phase capture and release methods were also demonstrated with unfractionated whole histone protein mixtures to show this compound applicability in real biological samples. The new chemical strategy will ultimately be utilized for high-throughput profiling of phosphorylation and possibly other posttranslational modifications.
Chowdhury
Saiful M
SM
Department of Chemistry, Washington State University, Pullman, WA 99164-4630, USA.
Munske
Gerhard R
GR
Siems
William F
WF
Bruce
James E
JE
D000818:Animals; D002364:Caseins / Q000737:chemistry; D045424:Complex Mixtures / Q000032:analysis; D006657:Histones / Q000032:analysis; D008301:Maleimides / Q000737:chemistry*; D008623:Mercaptoethanol / Q000031:analogs & derivatives* / Q000737:chemistry; D010047:Ovalbumin / Q000737:chemistry; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000032:analysis / Q000737:chemistry*; D010766:Phosphorylation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013438:Sulfhydryl Compounds / Q000737:chemistry
D002364:Caseins; D045424:Complex Mixtures; D006657:Histones; D008301:Maleimides; D010748:Phosphopeptides; D013438:Sulfhydryl Compounds; C030762:propanethiol; C043592:maleimide; D008623:Mercaptoethanol; D010047:Ovalbumin; C031854:1,2-ethanedithiol