Highly Efficient Phosphopeptide Enrichment by Calcium Phosphate Precipitation Combined with Subsequent IMAC Enrichment.
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.
Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense, Denmark.
Xumin
X
Zhang
Juanying
J
Ye
Ole N
ON
Jensen
Peter
P
Roepstorff
D000595:Amino Acid Sequence; D002130:Calcium Phosphates / Q000737:chemistry*; D011232:Chemical Precipitation; D002853:Chromatography, Liquid / Q000379:methods*; D007093:Imidazoles / Q000737:chemistry*; D007501:Iron / Q000737:chemistry; D008969:Molecular Sequence Data; D012275:Oryza / Q000737:chemistry; D010748:Phosphopeptides / Q000737:chemistry / Q000302:isolation & purification*; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014025:Titanium / Q000737:chemistry
D002130:Calcium Phosphates; D007093:Imidazoles; D010748:Phosphopeptides; D010940:Plant Proteins; C009495:titanium dioxide; C020243:calcium phosphate; D014025:Titanium; D007501:Iron; C005954:imidazoleacetic acid