Low-mass proteome analysis based on liquid chromatography fractionation, nanoliter protein concentration/digestion, and microspot matrix-assisted laser desorption ionization mass spectrometry.
HPLC fractionation combined with mass spectrometry can become a powerful tool for analyzing the proteome in the mass range below 15 kDa where efficient protein separation by gel electrophoresis can be difficult. For sensitive and high-resolution separation of the low-mass proteome, the use of analytical rather than preparative HPLC columns is preferred. However, individual fractions collected by a conventional HPLC separation usually contain a small amount of proteins whose concentrations may not be sufficiently high for subsequent enzyme digestion and protein identification by mass spectrometry. In this work, we present a high sensitivity nanoliter sample handling technique to analyze proteins fractionated by HPLC. In this technique, an individual HPLC fraction in hundreds of microliter volume is pre-concentrated to several microliters. About 700 pl of the pre-concentrated fraction is then drawn into a 20-microm I.D. capillary and dried in a small region near the capillary's entrance. This process can be repeated many times to concentrate a sufficient amount of protein to the small region of the capillary. After protein concentration, protein digestion is achieved by drawing 1 nl of chemical or enzymatic reagent into the capillary and placing it in the same region where the dried protein sits. The resulting peptides are then deposited onto a microspot in a MALDI probe for mass analysis. The performance of this technique is demonstrated with the use of a standard protein solution. This technique is applied to the identification of low-mass proteins separated by HPLC from a complex mixture of an E. coli extract.
Keller
Bernd O
BO
Department of Chemistry, Faculty of Science, University of Alberta, E3-44 Chemistry Building, Edmonton, Canada.
Wang
Zhengping
Z
Li
Liang
L
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D020543:Proteome; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D029968:Escherichia coli Proteins; D020543:Proteome