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
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