Protein Isoforms Observed by Ultrahigh Resolution Capillary Isoelectric Focusing electrospray Ionization Mass Spectrometry

LIU Tao, SHAO Xiao-Xia, ZENG Rong, XIA Qi-Chang^*
( Research Centre for Proteomic Analysis, Key Laboratory of Proteomics,Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, the Chinese Academy of Sciences,Shanghai 200031, China )

Abstract    On-line coupling of capillary isoelectric focusing (CIEF) to electrospray ionization mass spectrometry (ESI-MS) as a two-dimensional separation/analysis system was employed for high-resolution analysis of the protein isoforms observed during CIEF process. The analytical system was established by using neutral coated long capillary (80 cm), active capillary positioning and sheath-liquid interface. Proteins were separated and resolved in CIEF according to their differences in isoelectric point (pI), and then characterized by ESI-MS. The focused protein zones were eluted to the entrance of MS by combining cathodic mobilization with gravity. The ultrahigh resolution (difference in pI<0.04) of this technique obtained under certain conditions led to the detection of three isoforms in hemoglobin A and in sickle cell hemoglobin (with similar charge distribution and same molecular weight, but their differences in pIranging from 0.04 to 0.08) and two isoforms of β-lactoglobulin A (difference in pI is 0.6). The isoelectric points, relative amounts, and molecular masses of these isoforms were determined simultaneously by CIEF-ESI-MS.
Key words    capillary isoelectric focusing; electrospray ionization mass spectrometry; protein isoform; hemoglobin variants

^*Corresponding author: Tel, 86-21-64374430; Fax, 86-21-64338357; e-mail, [email protected]