Biomedical subjects

Bart de Koning

Publications and source records attributed to Bart de Koning.

3 recordsLinked to original sources

PubMed2007

Relative quantification of proteins across the species boundary through the use of shared peptides.

We show that shared peptides of proteins that are encoded in different species are suitable for cross-species relative protein quantification. A 14N-containing proteome from the thermoacidophilic archaeon Sulfolobus tokodaii was mixed with a 15N-labeled proteome from Sulfolobus solfataricus. Using three shared peptides per protein, the relative abundance of six orthologous proteins was calculated. Observed standard deviations were approximately 10%, indicating that the trypsin accessibility to cleavage sites was not altered in the orthologs. The abundance ratios of the and subunits of the Thermosome were 0.64 and 1.24 in Sulfolobus tokodaii compared to Sulfolobus solfataricus, suggesting a different stoichiometry of the complex in both species. In addition, an in silico study was performed on the occurrence of shared peptides. Inter- and intra-species peptide redundancy was investigated in the model organisms Homo sapiens, Mus musculus, Escherichia coli K12, Escherichia coli O157:H7, S. solfataricus, and S. tokodaii. M. musculus and H. sapiens share 30-50% of all peptides (6-15 residues). Moreover, approximately one-third of all proteins shared > or = 40% of their peptides with at least one other protein in the related species, thus offering strong potential for cross-species relative protein quantification. Conversely, approximately 40% of all peptides (6-15 residues) encoded in H. sapiens are encoded multiple times and therefore complicate identification and quantification.

Amino Acid Sequence↗

PubMed2005

A fast method for quantitative proteomics based on a combination between two-dimensional electrophoresis and 15N-metabolic labelling.

We provide a method for accurate protein quantitation that uses two-dimensional (2-D) gel electrophoresis for protein separation, but does not require extensive statistical analysis of staining intensities on gels. Instead, accurate quantitation occurs on the mass spectrometer (MAS) on multiple peptides to provide statistical evidence. In an example study, Sulfolobus solfataricus cells were grown on the carbon sources glucose, fructose and glutamate. The glucose phenotype (reference) was grown on (15)N-enriched medium. Next, the glutamate and the fructose phenotypes are mixed with the reference and two 2-D gels are created. Staining intensities of gel spots in this case are used for initial, semiquantitative assessment of differential expression. On this basis, spots are selected for accurate quantitation on the MAS. A number of differentially expressed proteins were found, for example: a (25.2 +/- 8.2)-fold upregulation of isocitrate lyase and a (7.14 +/- 0.82)-fold downregulation of glucose dehydrogenase on glutamate compared to glucose. With this protocol, intergel and interlaboratory comparisons are facilitated, since the light and heavy versions of a protein are equally affected by variations in sample preparation and buffer composition. Because the statistical evidence is gathered on the MAS, the need to run vast numbers of gels is removed.

Down-Regulation↗

PubMedDate not supplied

Perturbation and interpretation of nitrogen isotope distribution patterns in proteomics.

This study provides a discussion on the applications and limitations of (15)NH(4)(+) metabolic labeling in proteomic studies. The hyperthemophilic crenarchaeon Sulfolobus solfataricus was used as a model organism throughout this study. The distribution of nitrogen was studied in four different experiments in which this distribution was manipulated in a unique way. The experiments included full adaptation to media with relative isotope abundances (RIA) of 0.36%, 50%, and >98% (15)NH(4)(+). The incorporation efficiency was calculated on the basis of a comparison between theoretical and experimental spectra. In the case of full adaptation, incorporation efficiencies reflected the RIA (0.36%, 47.5% and 99% respectively). Labeling efficiencies were calculated on the basis of peak areas in TOF-MS spectra. It is shown that in the case of full adaptation, labeling efficiencies are 100%. In addition, we demonstrate that (15)NH(4)(+) labeling can be used in protein turnover studies, even when labeling is incomplete. In this case, incorporation efficiencies of 88-93% (lower than the RIA) were measured, providing evidence for amino acid recycling. Labeling efficiencies were always between 63% and 94% providing evidence for protein degradation. Finally, it was shown that isotope distributions can be useful in peptide identification.

Ammonia↗