Multiple, simultaneous, independent gradients for a versatile multidimensional liquid chromatography. Part II: Application 1 - Large increases in isoform resolution of human transferrin by use of dual simultaneous independent gradients of pH & acetonitrile on a mixed bed (anion exchange plus reversed phase) stationary phase.
We have previously described a liquid chromatographic (LC) method for uncoupling controlled, wide range pH gradients and simultaneous controlled gradients of a non-buffering solute on ion exchange resins (Hirsh and Tsonev, 2012) [1]. Here we report the application of this two dimensional LC technique to the problem of resolving Human Transferrin (HT) isoforms. This important iron transporting protein should theoretically occur in several thousand glycoforms, but only about a dozen have been reported. Using dual simultaneous independent gradients (DSIGs) of acetonitrile (ACN) and pH on a mixed bed stationary phase (SP) consisting of a mixture of an anion exchange resin and a reversed phase (RP) resin we partially resolve about 60 isoforms. These are likely to be partially refolded glycoforms generated by interaction of HT with the highly hydrophobic RP SP, as well as distinct folded glycoforms. Thus this study should have interesting implications for both glycoform separation and the study of protein folding.
CryoBioPhysica, Inc., 4620 N. Park Ave., #1204E, Chevy Chase, MD 20815, United States.
Latchezar I
LI
Tsonev
CryoBioPhysica, Inc., 4620 N. Park Ave., #1204E, Chevy Chase, MD 20815, United States. Electronic address: agh@cryobiophysica.com.
Allen G
AG
Hirsh
D000097:Acetonitriles; D000837:Anion Exchange Resins; D002852:Chromatography, Ion Exchange / Q000379:methods; D002853:Chromatography, Liquid; D006801:Humans; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D017510:Protein Folding; D020033:Protein Isoforms / Q000302:isolation & purification; D014168:Transferrin / Q000302:isolation & purification*
D000097:Acetonitriles; D000837:Anion Exchange Resins; D020033:Protein Isoforms; D014168:Transferrin; C032159:acetonitrile
Human transferrin isoforms; Mixed bed stationary phases; Multi-dimensional liquid chromatography; Protein chromatography; Protein folding intermediates; pH gradient chromatography