Top-down analysis of basic proteins by microchip capillary electrophoresis mass spectrometry.
A system of microchip capillary electrophoresis/electrospray ionization mass spectrometry (microchip-CE/ESI-MS) for rapid characterization of proteins has been developed. Capillary electrophoresis (CE) enables rapid analysis of a sample present in very small quantity, such as at femtomole levels, at high resolution. Faster CE/MS analysis is expected by downsizing the normal capillary to the microchip (microchip) capillary. Although rapidity and high resolution are advantages of CE separation, electroosmotic flow (EOF) instability caused by the interaction between proteins and the microchannel surface results in low reproducibility in the analysis of basic proteins under neutral pH conditions. By coating the microchannel surface with a basic polymer, polyE-323, basic proteins, which have pI values of over 7.5, could be separated and detected by microchip-CE/MS on quadrupole (Q) and time-of-flight (TOF) hybrid instruments. By increasing the cone and collision voltages during the analysis by microchip-CE/ESI-MS of a small protein, some product ions, which contain the sequence information, could also be obtained, i.e., 'top-down' analysis of the protein could be accomplished with this microchip-CE/MS system. To our knowledge, this is the first report of 'top-down' analysis of a protein by microchip-CE/MS. Since it requires a much shorter time and a smaller sample amount for analysis than the conventional liquid chromatography (LC)/ESI-MS method, microchip-CE/MS promises to be suitable for the high-throughput characterization of proteins.
Akashi
Satoko
S
International Graduate School of Arts and Sciences, Yokohama City University, 1-7-29 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan. akashi@tsurumi.yokohama-cu.ac.jp
Suzuki
Koichi
K
Arai
Akihiro
A
Yamada
Naoyuki
N
Suzuki
Ei-Ichiro
E
Hirayama
Kazuo
K
Nakamura
Shin
S
Nishimura
Yoshifumi
Y
D000818:Animals; D002645:Chickens; D046209:Electrophoresis, Microchip / Q000379:methods*; D004743:Enkephalin, Leucine / Q000032:analysis; D006736:Horses; D006801:Humans; D008832:Microchemistry / Q000295:instrumentation / Q000379:methods; D009113:Muramidase / Q000032:analysis; D009211:Myoglobin / Q000032:analysis; D009687:Nuclear Proteins / Q000032:analysis; D011506:Proteins / Q000032:analysis*; D020539:Sequence Analysis, Protein / Q000295:instrumentation / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation / Q000379:methods*; D035182:TATA Box Binding Protein-Like Proteins / Q000032:analysis; D035341:Telomeric Repeat Binding Protein 2; D013997:Time Factors
D009211:Myoglobin; D009687:Nuclear Proteins; D011506:Proteins; D035182:TATA Box Binding Protein-Like Proteins; C504753:TERF2 protein, human; D035341:Telomeric Repeat Binding Protein 2; D004743:Enkephalin, Leucine; D009113:Muramidase