Automated multi-dimensional liquid chromatography: sample preparation and identification of peptides from human blood filtrate.
A comprehensive on-line sample clean-up with an integrated two-dimensional HPLC system was developed for the analysis of natural peptides. Samples comprised of endogenous peptides with molecular weights up to 20 kDa were generated from human hemofiltrate (HF) obtained from patients with chronic renal failure. The (poly-)peptides were separated using novel silica-based restricted access materials with strong cation-exchange functionalities (SCX-RAM). The size-selective sample fractionation step is followed by cation-exchange chromatography as the first dimension. The subsequent second dimension of separation is based on hydrophobic interaction using four parallel short reversed-phase (RP) columns implemented via a fully automated column switching technique. More than 1000 peaks were resolved within the total analysis time of 96 min. Substances of selected peaks were sampled to analyse their molecular weights by off-line MALDI-TOF mass spectrometry and to determine their amino acid sequence by Edman degradation. The potential for comprehensive peptide mapping and identification is demonstrated.
Machtejevas
Egidijus
E
Institut fuer Anorganische Chemie und Analytische Chemie, Johannes Gutenberg-Universitaet, Duesbergweg 10-14, D-55099 Mainz, Germany. egidijus@ak-unger.chemie.uni-mainz.de
John
Harald
H
Wagner
Knut
K
Ständker
Ludger
L
Marko-Varga
György
G
Forssmann
Wolf-Georg
WG
Bischoff
Rainer
R
Unger
Klaus K
KK
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002852:Chromatography, Ion Exchange; D005374:Filtration; D006801:Humans; D008969:Molecular Sequence Data; D010455:Peptides / Q000097:blood* / Q000737:chemistry
D010455:Peptides