Rapid protein identification using a disposable on-line clean-up/concentrating device and electrospray ionization mass spectrometry

• Published in: Rapid communications in mass spectrometry 2007-01, Vol.21 (4), p.459-465
• Main Authors: Tsai, Chu-Yun, Pai, Pei-Jing, Ho, Yi-Hsin, Lu, Jyh-Feng, Wang, Jinn-Shyan, Lin, Wann-Yin, Her, Guor-Rong
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Cattle; Electrophoresis, Gel, Two-Dimensional; Molecular Sequence Data; Myoglobin - chemistry; Peptide Mapping - methods; Peptides - analysis; Serum Albumin, Bovine - chemistry; Software; Spectrometry, Mass, Electrospray Ionization - methods
• ISSN: 0951-4198; 1097-0231
• DOI: 10.1002/rcm.2857

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 µm 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 (µLC/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. Copyright © 2007 John Wiley & Sons, Ltd.