Biomedical subjects

Dan Bylund

Publications and source records attributed to Dan Bylund.

4 recordsLinked to original sources

PubMed2003

Analysis of phosphatidylcholine and sphingomyelin molecular species from brain extracts using capillary liquid chromatography electrospray ionization mass spectrometry.

One feature of complex lipids is that many subtypes of these molecules exist as a diverse mixture in a biological sample. Qualitative and quantitative analysis of these closely related molecules require sensitive and specific analytical methods to detect intact phospholipids (PL) and sphingomyelin (SM) species and to differentiate between them. Conventional analytical methods require laborious procedures including separation by column, argentation thin-layer chromatography or liquid chromatography (LC) after pre- or post-column derivatization. In the present work, a method based on reversed phase capillary LC coupled on-line to electrospray ionization mass spectrometry (LC/ESI/MS) has been developed to gather tools for lipidomic studies, i.e. the profiling of complex mixtures of lipids in small amounts of various cells and tissues. The LC/MS system used consisted of an LC pump in an isocratic elution, a reversed phase capillary column and a single quadrupole mass spectrometer operated in the positive ion mode. A successful separation of phosphatidylcholine (PC) and SM molecular species was obtained with a minimum detectable quantity (MDQ) in the low fmol range injected on column. The method was applied to human brain extracts. Furthermore, the extraction efficiencies of the traditional Folch method and pressurized fluid extraction (PFE) were compared using the human brain. It was found that the intensity of the PC and SM molecular species extracted by PFE is two times that of Folch.

Aged↗

PubMed2003

Miniaturized on-line proteolysis-capillary liquid chromatography-mass spectrometry for peptide mapping of lactate dehydrogenase.

In this study, methodology was developed for on-line and miniaturized enzymatic digestion with liquid chromatographic (LC) separation and mass spectrometric (MS) detection. A packed capillary LC-MS system was combined with on-line trypsin cleavage of a model protein, lactate dehydrogenase, to provide an efficient system for peptide mapping. The protein was injected onto an enzymatic capillary reactor and the resulting peptides were efficiently trapped on a capillary trapping column. Different trapping columns were evaluated to achieve a high binding capacity for the peptides generated in the enzyme reactor. The peptides were further eluted from the pre-column and separated on an analytical capillary column by a buffer more suitable for the following an electrospray ionisation (ESI) MS process. An important aspect of the on-line approach was the desalting of peptides performed in the trapping column to avoid detrimental signal suppression in the ESI process. The developed on-line system was finally compared to a classical digestion in solution, with reference to peptide sequence coverage and sensitivity. It was shown that the on-line system gave more than 100% higher peptide sequence coverage than traditional digestion methods.

Animals↗

PubMed2003

Classification of lactate dehydrogenase of different origin by liquid chromatography-mass spectrometry and multivariate analysis.

A method intended to serve as a multivariate quality control tool in the production of pharmaceutical proteins is presented. The method is based on multivariate analysis of peptide maps generated with liquid chromatography-mass spectrometry (LC-MS). Lactate dehydrogenase (LDH) from different species and tissues were used as model compounds in the study. The proteins were digested with Endoproteinase Lys-C before the LC-MS analysis. After data pretreatment of the peptide maps, successful classification of the LDHs were obtained by discriminant analysis with partial least squares regression and artificial neural networks. Further, principal component analysis was applied to visualize the relationships between the samples.

Animals↗

PubMed2002

Chromatographic alignment by warping and dynamic programming as a pre-processing tool for PARAFAC modelling of liquid chromatography-mass spectrometry data.

Solutes analysed with LC-MS are characterised by their retention times and mass spectra, and quantified by the intensities measured. This highly selective information can be extracted by multiway modelling. However, for full use and interpretability it is necessary that the assumptions made for the model are valid. For PARAFAC modelling, the assumption is a trilinear data structure. With LC-MS, several factors, e.g. non-linear detector response and ionisation suppression may introduce deviations from trilinearity. The single largest problem, however, is the retention time shifts not related to the true sample variations. In this paper, a time warping algorithm for alignment of LC-MS data in the chromatographic direction has been examined. Several refinements have been implemented and the features are demonstrated for both simulated and real data. With moderate time shifts present in the data, pre-processing with this algorithm yields approximately trilinear data for which reasonable models can be made.

Chromatography, Liquid↗