High resolution analysis of protein phosphorylation using capillary isoelectric focusing - electrospray ionization - mass spectrometry.
On-line capillary isoelectric focusing (CIEF)-electrospray ionization - mass spectrometry (ESI-MS) as a two-dimensional separation system is employed for high resolution analysis of ovalbumin phosphorylation. On the basis of their differences in isoelectric point (pI), the mono- and diphosphoovalbumins are separated and resolved in CIEF. The focused protein zones of mono- and diphosphoovalbumins are eluted by combining gravity with cathodic mobilization. At the end of the CIEF capillary, the mobilized ovalbumin zones are analyzed by mass spectrometry coupled on-line to an electrospray interface with a coaxial sheath flow configuration. Additional ovalbumin variants within each of the mono- and diphosphoovalbumins, differing in their molecular masses due to glycosylation microheterogeneity, are easily distinguished by ESI-MS.
Wei
J
J
Department of Chemistry and Ames Laboratory, Iowa State University, USA.
Yang
L
L
Harrata
A K
AK
Lee
C S
CS
D019075:Electrophoresis, Capillary / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing / Q000379:methods*; D007526:Isoelectric Point; D009682:Magnetic Resonance Spectroscopy; D008358:Mannose / Q000032:analysis; D013058:Mass Spectrometry / Q000379:methods*; D008970:Molecular Weight; D010047:Ovalbumin / Q000302:isolation & purification / Q000378:metabolism*; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010766:Phosphorylation
D010750:Phosphoproteins; D010047:Ovalbumin; D010744:Phosphoric Monoester Hydrolases; D008358:Mannose