Miniaturized Techniques for Protein Analysis
Proteins are a highly diversified group of molecules, andfor their study, advanced analytical tools are required. Inparticular, a need for high-throughput techniques has emergedin order to enable the characterization of large sets ofproteins. In this thesis, improved techniques for proteinseparation...
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KTH, Kemi
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ftkthstockholm:oai:DiVA.org:kth-3802 2024-06-23T07:47:33+00:00 Miniaturized Techniques for Protein Analysis Sjödahl, Johan 2004 application/pdf http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-3802 eng eng KTH, Kemi Stockholm : Kemi http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-3802 urn:isbn:91-7283-783-7 info:eu-repo/semantics/openAccess Proteins peptides proteases Antarctic krill Euphausia superba capillary electrophoresis fluorosurfactants mass spectrometry nanoelectrospray ESI MALDI chip high-throughput reproducibility sensitivity mass accuracy Doctoral thesis, comprehensive summary info:eu-repo/semantics/doctoralThesis text 2004 ftkthstockholm 2024-05-27T17:41:32Z Proteins are a highly diversified group of molecules, andfor their study, advanced analytical tools are required. Inparticular, a need for high-throughput techniques has emergedin order to enable the characterization of large sets ofproteins. In this thesis, improved techniques for proteinseparations as well as new tools for the mass spectrometricanalysis of proteins are described. In the work, presented in the first part of the thesis, arefined extract containing proteases from Antarctic krill (Euphausia superba) was separated and characterized bymeans of capillary electrophoresis (CE) and mass spectrometry(MS). Tailored CE separations of the krill extract revealed thepresence of approximately 50 components. In addition, adetailed CE and MS analysis of fractions, containing individualkrill proteases has been carried out. Trypsin-like proteasesfrom krill exhibited a 12-fold and a 60-fold higher digestionefficiency at 37 °C and 2 °C respectively compared todigests performed with bovine trypsin. Furthermore, thecleavage specificity of the trypsin-like proteases wasstudied. In the last part of the thesis, novel concepts forchip-based nanoelectrospray (nanoESI) and matrix-assisted laserdesorption/ionization (MALDI) mass spectrometry are described.First, a micromachined silicon chip with a two-dimensionalmatrix of out-ofplane nanoESI needles for high-throughputanalysis was fabricated. A two-fold improvement insignal-to-noise reproducibility was obtained. Second, achip-based target for MALDI was developed, which featured pairsof elevated 50×50 µm anchors in close proximity. Theanchors were individually addressable with sample solution. Theminiaturized sample preparations at close distance to eachother allowed a simultaneous ionization of a physicallyseparated sample and standard by one single laser pulse. Thisresulted in a twofold reduction of relative mass errors.Moreover, ion suppression of the analyte was significantlyreduced. The effective utilization of the sample resulted in adetection limit of ca 200 ... Doctoral or Postdoctoral Thesis Antarc* Antarctic Antarctic Krill Euphausia superba Royal Institute of Technology, Stockholm: KTHs Publication Database DiVA Antarctic |
institution |
Open Polar |
collection |
Royal Institute of Technology, Stockholm: KTHs Publication Database DiVA |
op_collection_id |
ftkthstockholm |
language |
English |
topic |
Proteins peptides proteases Antarctic krill Euphausia superba capillary electrophoresis fluorosurfactants mass spectrometry nanoelectrospray ESI MALDI chip high-throughput reproducibility sensitivity mass accuracy |
spellingShingle |
Proteins peptides proteases Antarctic krill Euphausia superba capillary electrophoresis fluorosurfactants mass spectrometry nanoelectrospray ESI MALDI chip high-throughput reproducibility sensitivity mass accuracy Sjödahl, Johan Miniaturized Techniques for Protein Analysis |
topic_facet |
Proteins peptides proteases Antarctic krill Euphausia superba capillary electrophoresis fluorosurfactants mass spectrometry nanoelectrospray ESI MALDI chip high-throughput reproducibility sensitivity mass accuracy |
description |
Proteins are a highly diversified group of molecules, andfor their study, advanced analytical tools are required. Inparticular, a need for high-throughput techniques has emergedin order to enable the characterization of large sets ofproteins. In this thesis, improved techniques for proteinseparations as well as new tools for the mass spectrometricanalysis of proteins are described. In the work, presented in the first part of the thesis, arefined extract containing proteases from Antarctic krill (Euphausia superba) was separated and characterized bymeans of capillary electrophoresis (CE) and mass spectrometry(MS). Tailored CE separations of the krill extract revealed thepresence of approximately 50 components. In addition, adetailed CE and MS analysis of fractions, containing individualkrill proteases has been carried out. Trypsin-like proteasesfrom krill exhibited a 12-fold and a 60-fold higher digestionefficiency at 37 °C and 2 °C respectively compared todigests performed with bovine trypsin. Furthermore, thecleavage specificity of the trypsin-like proteases wasstudied. In the last part of the thesis, novel concepts forchip-based nanoelectrospray (nanoESI) and matrix-assisted laserdesorption/ionization (MALDI) mass spectrometry are described.First, a micromachined silicon chip with a two-dimensionalmatrix of out-ofplane nanoESI needles for high-throughputanalysis was fabricated. A two-fold improvement insignal-to-noise reproducibility was obtained. Second, achip-based target for MALDI was developed, which featured pairsof elevated 50×50 µm anchors in close proximity. Theanchors were individually addressable with sample solution. Theminiaturized sample preparations at close distance to eachother allowed a simultaneous ionization of a physicallyseparated sample and standard by one single laser pulse. Thisresulted in a twofold reduction of relative mass errors.Moreover, ion suppression of the analyte was significantlyreduced. The effective utilization of the sample resulted in adetection limit of ca 200 ... |
format |
Doctoral or Postdoctoral Thesis |
author |
Sjödahl, Johan |
author_facet |
Sjödahl, Johan |
author_sort |
Sjödahl, Johan |
title |
Miniaturized Techniques for Protein Analysis |
title_short |
Miniaturized Techniques for Protein Analysis |
title_full |
Miniaturized Techniques for Protein Analysis |
title_fullStr |
Miniaturized Techniques for Protein Analysis |
title_full_unstemmed |
Miniaturized Techniques for Protein Analysis |
title_sort |
miniaturized techniques for protein analysis |
publisher |
KTH, Kemi |
publishDate |
2004 |
url |
http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-3802 |
geographic |
Antarctic |
geographic_facet |
Antarctic |
genre |
Antarc* Antarctic Antarctic Krill Euphausia superba |
genre_facet |
Antarc* Antarctic Antarctic Krill Euphausia superba |
op_relation |
http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-3802 urn:isbn:91-7283-783-7 |
op_rights |
info:eu-repo/semantics/openAccess |
_version_ |
1802651675901558784 |