Protein isoforms observed by ultrahigh resolution capillary isoelectric focusing electrospray ionization mass spectrometry.
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 beta-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.
Liu
Tao
T
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. xiaqc@sunm.shcnc.ac.cn
Shao
Xiao-Xia
XX
Zeng
Rong
R
Xia
Qi-Chang
QC
D024402:Carbonic Anhydrase II / Q000032:analysis; D003574:Cytochrome c Group / Q000032:analysis; D006441:Hemoglobin A / Q000032:analysis; D006451:Hemoglobin, Sickle / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing / Q000379:methods*; D007782:Lactoglobulins / Q000032:analysis; D009211:Myoglobin / Q000032:analysis; D020033:Protein Isoforms / Q000032:analysis; D011506:Proteins / Q000032:analysis*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D003574:Cytochrome c Group; D006451:Hemoglobin, Sickle; D007782:Lactoglobulins; D009211:Myoglobin; D020033:Protein Isoforms; D011506:Proteins; D006441:Hemoglobin A; D024402:Carbonic Anhydrase II