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- Grimm, C. H., et al.
(författare)
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Selective extraction of small proteins from biological samples using a novel restricted access column with cation exchange properties
- 2000
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Ingår i: Chromatographia. - 0009-5893. ; 52:11-12, s. 703-709
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Tidskriftsartikel (refereegranskat)abstract
- The determination of proteins utilising a polymer-based restricted access support material with ion exchange properties (IERAM) is outlined. Solid phase extraction coupled on-line with a microbore reversed phase HPLC system for the quantitation of small marker proteins is demonstrated. The cation-exchange restricted access packings were characterised with respect to their adsorption and desorption kinetics. The IERAM material was also investigated by capacity, selectivity, and biocompatibility determinations when applied to the quantification of small molecular weight proteins such as cytochrome C, Lysozyme, Ribonuclease A, Myoglobin, Insulin, human serum albumin, and a Tryptic inhibitor. The integrated system was coupled to mass identity of selected proteins by MALDI-TOF mass spectrometry. The chromatographic outlet was interfaced to an "Interplate" fractionation collecter that sampled 1 μL volumes directly onto the MALDI target plate. The fully automated coupled column system was run unattended overnight and applied to protein quantitations in human plasma samples. Recovery data for a selected number of proteins varied between 90-96% (n = 10) with a limit of quantification around 2 μM with an injection volume of 100 μL. The RSD data were typically less than 8% at a 50 μM protein level (n = 7).
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| 2. |
- Machtejevas, E, et al.
(författare)
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Automated multi-dimensional liquid chromatography: sample preparation and identification of peptides from human blood filtrate
- 2004
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Ingår i: Journal of Chromatography. B. - : Elsevier BV. - 1873-376X .- 1570-0232. ; 803:1, s. 121-130
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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