Gradient chromatofocusing-mass spectrometry: a new technique in protein analysis.
A new analytical technique, gradient chromatofocusing-mass spectrometry (gCF-MS), was developed employing ion-exchange high-performance liquid chromatography (HPLC) interfaced to an electrospray-quadrupole mass spectrometer in the determination of proteins. There have been few reports, if any, of a HPLC-MS technique for proteins in which the ion-exchange column is directly interfaced to the mass spectrometer. The employment of a linear pH gradient elution scheme directly interfaced to mass spectrometry is also unique in the present work. The technique was demonstrated by the separation of six proteins (carbonic anhydrase II, enolase, beta-lactoglobulin A, lactoglobulin B, soybean trypsin inhibitor, and amyloglucosidase) employing a descending linear pH gradient from pH 9 to 2.6 on a 50 mm x 2.1 mm DEAE HPLC column using volatile buffer components. A signal enhancement solution consisting of 8% formic acid in acetonitrile was pumped post-column and was mixed 1:1 with column effluent and then directed on-line into the mass spectrometer. Molecular masses of the proteins were determined within +/-0.010% to 0.033% (+/-100 to 330 ppm) with peak height total ion current detection limits of 4 to 78 pmol of injected amounts (S/N = 3). This technique is applicable to the analysis of proteins and other charged molecules.
Shan
Lian
L
Department of Chemistry, Cleveland State University, 2121 Euclid Avenue, Cleveland, OH 44115, USA.
Hribar
James A
JA
Zhou
Xiang
X
Anderson
David J
DJ
D002021:Buffers; D002845:Chromatography; D002848:Chromatography, DEAE-Cellulose; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003627:Data Interpretation, Statistical; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry / Q000379:methods*; D011506:Proteins / Q000737:chemistry*; D012492:Salts / Q000737:chemistry; D012996:Solutions
D002021:Buffers; D011506:Proteins; D012492:Salts; D012996:Solutions