Abstract
A new method for enrichment of phosphopeptides in complex mixtures derived by proteolytic digestion of biological samples has been developed. The method is based on calcium phosphate precipitation of the phosphopeptides prior to further enrichment with established affinity enrichment methods. Calcium phosphate precipitation combined with phosphopeptide enrichment using Fe(III) IMAC provided highly selective enrichment of phosphopeptides. Application of the method to a complex peptide sample derived from rice embryo resulted in more than 90% phosphopeptides in the enriched sample as determined by mass spectrometry. Introduction of a two-step IMAC enrichment procedure after calcium phosphate precipitation resulted in observation of an increased number of phosphopeptides.
Highlights
A new method for enrichment of phosphopeptides in complex mixtures derived by proteolytic digestion of biological samples has been developed
We decided to use ␣- and -casein as test phosphoproteins because these proteins have been extensively used as models in the development of methods for phosphoproteomics (18, 20, 40) due to their multiple phosphorylated sites (Table I)
A method for phosphopeptide enrichment based on calcium phosphate precipitation has been developed
Summary
A new method for enrichment of phosphopeptides in complex mixtures derived by proteolytic digestion of biological samples has been developed. Calcium phosphate precipitation combined with phosphopeptide enrichment using Fe(III) IMAC provided highly selective enrichment of phosphopeptides. Application of the method to a complex peptide sample derived from rice embryo resulted in more than 90% phosphopeptides in the enriched sample as determined by mass spectrometry. Mass spectrometry is one of the most powerful techniques for protein identification, and it is playing an increasingly important role in characterization of post-translational modifications, including protein phosphorylation (6). Chromatographic enrichment of the phosphopeptides has been widely used in phosphoproteome studies This includes IMAC (17, 19 –21), strong cation exchange chromatography (22, 23), strong anion exchange chromatography (24), and metal oxide chromatography (18, 25–27). Application of the new method of calcium phosphate precipitation and IMAC to studies of the rice embryo phosphoproteome demonstrated that a highly selective enrichment of phosphopeptides was achieved in very complex peptide samples
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
