Johan Sjödahl

Development of micromachined hollow tips for protein analysis based on nanoelectrospray ionization mass spectrometry

Two novel types of micromachined nanoelectrospray emitter tips have been designed, fabricated and tested. The fabrication method of the hollow tips is based on a self-aligning deep reactive ion etch process. The tips consist of either silicon dioxide or silicon and feature orifice diameters of 10 and 18 μm, respectively. The geometrical characteristics of both emitter types are favorable for the generation of stable electrospray ionization, i.e. wetting of the tip shaft is avoided and the base of the Taylor cone is limited to the diameter of the orifice. A silicon dioxide tip was operated in a bench top setup to visually evaluate the electrospray. Both types of tips were also successfully used for the analysis of an insulin sample in an ion trap mass spectrometer.

Characterization of proteinases from Antarctic krill (Euphausia superba)

Fractions of three trypsin-like proteinases, TL I, TL II, and TL III, a chymotrypsin-like proteinase, CL, two carboxypeptidase A enzymes, CPA I and CPA II and two carboxypeptidase B enzymes, CPB I and CPB II, from Antarctic krill (Euphausia superba) have been characterized with respect to purity by the means of capillary electrophoresis, CE, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The masses of the trypsin-like and chymotrypsin-like proteinases were determined to be 25,020, 25,070, 25,060, and 26,260Da for TL I, TL II, TL III, and CL, respectively. The masses of the CPA enzymes are likely 23,170 and 23,260Da, whereas the CPB enzyme masses likely are 33,730 and 33,900Da. The degradation efficiency and cleavage pattern of the trypsin-like proteinases were studied with native myoglobin as a model substrate using CE, MALDI-TOF-MS, and nanoelectrospray mass spectrometry (nESI-MS). The degradation efficiency of the trypsin-like proteinases was found to be approximately 12 and 60 times higher compared to bovine trypsin at 37 degrees C and 1-3 degrees C, respectively. All three fractions of trypsin-like proteinases showed a carboxypeptidase activity in combination with their trypsin activity.

Separation of proteolytic enzymes originating from Antarctic krill (Euphausia superba) by capillary electrophoresis

Extracts prepared from Antarctic krill (Euphausia superba), mainly consisting of acidic proteolytic enzymes, have been studied with capillary electrophoretic techniques. Approximately 50 repeatable peaks were obtained with capillary zone electrophoresis on an untreated fused-silica capillary using a phosphate buffer containing anionic and cationic fluorosurfactant additives as separation medium. A faster separation was achieved on a polyvinyl alcohol coated capillary. Quantitative variations of individual proteins regarding different krill enzyme batches were noted. In the krill samples trypsin-like serine proteinase, carboxypeptidase A and carboxypeptidase B were tentatively identified.