Characterization of proteins in the human serum proteome.
This paper presents a multidimensional profile of the human serum proteome, produced by a two-dimensional protein fractionation system based on liquid chromatography followed by characterization with capillary electrophoresis (CE). The first-dimension separation was done by chromatofocusing over a pH range from 8.5 to 4.0, where proteins were separated by their isoelectric points (pI). In this dimension, fractions were collected based on pH. The first-dimension pI fractions were then resolved in the second dimension by high-resolution, reversed-phase chromatography with a gradient of trifluoroacetic acid (TFA) in acetonitrile and TFA in water. A selected protein fraction collected from the second dimension by time was characterized by CE for molecular-weight estimation and for presence of isoforms. Molecular-weight estimation was done by sodium dodecyl sulfate capillary gel electrophoresis, where proteins were separated in the range of 10,000-225,000 Da. Detection of isoforms was done by capillary isoelectric focusing over a pH range of 3-10. A selected second-dimension fraction that contained the putative serum iron-binding protein transferrin was analyzed by these two CE techniques for molecular-weight determination and the presence of isoforms. The combination of two-dimensional protein fractionation and CE characterization represents an advanced tool for proteomics.
Betgovargez
Edna
E
Biomedical Research Division, Beckman Coulter, Inc., Fullerton, California, USA. ebetgovargez@beckman.com
Knudson
Vita
V
Simonian
Michael H
MH
D000328:Adult; D001798:Blood Proteins / Q000032:analysis* / Q000302:isolation & purification*; D002853:Chromatography, Liquid; D019075:Electrophoresis, Capillary; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D008297:Male; D008970:Molecular Weight; D020033:Protein Isoforms / Q000737:chemistry; D020543:Proteome / Q000032:analysis* / Q000302:isolation & purification*; D040901:Proteomics; D012015:Reference Standards
D001798:Blood Proteins; D020033:Protein Isoforms; D020543:Proteome