Biomedical subjects
Publications and source records attributed to Dariusz J Janecki.
A chemical derivatization method, amidination, that has recently been effectively employed in peptide mass spectrometry experiments is used to covalently modify lysines in several standard proteins. Protein and peptide mass spectra identify sites at which the reaction does or does not occur. This is therefore a rapid approach to elucidate solvent-accessible regions of folded proteins.
Metal ions bound to a protein often stabilize tertiary and/or quaternary structure. Consequently, the digestion of metalloproteins that precedes analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry is frequently incomplete. It is demonstrated that ethylenediaminetetraacetic acid (EDTA) successfully destabilizes metalloprotein structure and thereby facilitates tryptic digestion and protein identification.
Affinity capture surfaces can be prepared in a number of ways. A method of obtaining such surfaces through UV-activated immobilization of binding proteins using a benzophenone derivative is reported. Photoimmobilized protein G was used to selectively capture and preconcentrate bovine IgG from a mixture with BSA, and the affinity of photoattached concanavalin A toward ovalbumin was compared with that of commercially available concanavalin A on agarose beads. The results of the capture after tryptic digestion were analyzed by MALDI TOF MS. Immobilized trypsin was also prepared through photoimmobilization and later used to digest hemoglobin. Immobilized enzyme digestion resulted in more partial cleavages than solution-phase digestion. More methionine and tryptophan oxidation was also observed. Photoimmobilization was shown to be a quick and easy way of immobilizing ligands on surfaces.
Trypsin autolysis fragments and matrix clusters are often observed as intense peaks in mass spectra of protein digests. It is demonstrated that these can be exploited to improve the mass calibration of a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) spectrometer. Interpretation of some of the autolysis masses is complicated by the existence of disulfide bonds. Surprisingly large matrix clusters are often visible for alpha-cyano-4-hydroxy-cinnamic acid. The fractional part of their masses differentiates them from protein digestion fragments.