Rapid protein identification using a disposable on-line clean-up/concentrating device and electrospray ionization mass spectrometry.
A simple, low-cost, expedient method has been developed for identification of proteins isolated from two-dimensional (2D) gels. The method described uses a disposable on-line clean-up device, a syringe infusion pump and electrospray ionization mass spectrometry (ESI-MS). The on-line clean-up and concentrating device is a tapered capillary column filled with 1.5 cm of 5 microm C18 particles. The short column was easily prepared and was connected directly to the ESI source through a low-flow ESI sprayer. Peptides resulting from enzymatic digestion of proteins were eluted from the short column isocratically using a syringe infusion pump and analyzed by ESI-MS. This simple set-up was found useful in the analysis of proteins isolated from 2D gels. Compared to the more conventional micro-liquid chromatography/tandem mass spectrometry (microLC/MS/MS), this method can identify proteins rapidly without the need for an HPLC pump and removes the problem of cross-contamination caused by system carryover. These advantages make the method described competitive with conventional LC/MS even though the latter method gives slightly expanded sequence coverage.
Tsai
Chu-Yun
CY
Department of Chemistry, National Taiwan University, Taipei 106, Taiwan, ROC.
Pai
Pei-Jing
PJ
Ho
Yi-Hsin
YH
Lu
Jyh-Feng
JF
Wang
Jinn-Shyan
JS
Lin
Wann-Yin
WY
Her
Guor-Rong
GR
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D015180:Electrophoresis, Gel, Two-Dimensional; D008969:Molecular Sequence Data; D009211:Myoglobin / Q000737:chemistry*; D010449:Peptide Mapping / Q000379:methods*; D010455:Peptides / Q000032:analysis*; D012710:Serum Albumin, Bovine / Q000737:chemistry*; D012984:Software; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D009211:Myoglobin; D010455:Peptides; D012710:Serum Albumin, Bovine