Nanogram scale separations of proteins using capillary high-performance liquid chromatography with fully-automated on-line microfraction collection followed by matrix-assisted laser desorption ionisation time-of-flight mass spectrometry, protein sequencing and western blot analysis.
Capillary HPLC was applied for highly sensitive protein separations on a nanogram scale. A crude extract of acid soluble proteins from maize kernels was used as a model extract and separated on a 300-micron I.D. reversed-phase capillary column. Protein fractions of 1-4 microliters volume were fully automatically collected with a new robot microfraction collection system. Fraction collection was performed onto matrix assisted laser desorption ionisation time-of-flight targets for mass spectrometric analysis, onto sequencing membranes for automated Edman degradation and onto nitrocellulose membranes for Western blot analysis.
Grimm
R
R
Hewlett-Packard, Chemical Analysis Group Europe, Waldbronn, Germany.
Grasser
K D
KD
D000595:Amino Acid Sequence; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006023:Glycoproteins / Q000032:analysis / Q000737:chemistry; D006609:High Mobility Group Proteins / Q000032:analysis / Q000737:chemistry; D008969:Molecular Sequence Data; D009862:Online Systems; D010455:Peptides / Q000032:analysis* / Q000737:chemistry; D010940:Plant Proteins / Q000737:chemistry*; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014452:Ubiquitins / Q000032:analysis / Q000737:chemistry; D003313:Zea mays / Q000737:chemistry*
D006023:Glycoproteins; C106684:HMGa protein, Zea mays; D006609:High Mobility Group Proteins; D010455:Peptides; D010940:Plant Proteins; D014452:Ubiquitins; C005488:extensin protein, plant