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Mass Spectrometry Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 346 Managing and Exploring Large Data Sets Generated by Liquid Separation - Mass Spectrometry DANIEL BÄCKSTRÖM ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6214 UPPSALA ISBN 978-91-554-6976-4 2007 urn:nbn:se:uu:diva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apers included in this thesis This thesis is based on the following Papers, which are referred to in the text by their Roman numerals as indicated below: I. Nina Johannesson, Louise Olsson, Daniel Bäckström, Mag- nus Wetterhall, Rolf Danielsson, Jonas Bergquist. Screening for biomarkers in plasma from patients with gangrenous phlegmonous appendicitis using CE and CEC in combination with MS. Electrophoresis, 2007, 28, 1435-1443. II. Rolf Danielsson, Daniel Bäckström, Sara Ullsten. Rapid multivariate analysis of LC/GC/CE data (single or multiple channel detection) without prior peak alignment. Chemomet- rics and Intelligent Laboratory Systems, 2006, 84, 33-39. III. Sara Ullsten, Rolf Danielsson, Daniel Bäckström, Per Sjöberg, Jonas Bergquist. Urine profiling using capillary electrophoresis-mass spectrometry and multivariate data analysis. Journal of Chromatography A, 2006, 1117, 87-93. IV. Daniel Bäckström, My Moberg, Per Sjöberg, Jonas Ber- gquist, Rolf Danielsson. Multivariate comparison between peptide mass fingerprints obtained by Liquid Chromatgraphy- Electrospray Ionization Mass Spectrometry with different trypsin digestion procedures. Journal of Chromatography A, submitted. V. Daniel Bäckström, Erik Allard, Rolf Danielsson, Per Sjöberg, Jonas Bergquist. Comparing CE-MS fingerprints of urine samples, obtained after intake of coffee, tea or water. Manuscript. Reprints where made with kind permission from the publishers Author Contribution In Papers I-V, I was responsible for the data management including system maintenance and code implementation. In Paper I, I also assisted in the data evaluation and the writing of the Paper. In Paper II and III, I took part in the development of algorithms and their implementation, as well as evaluation of the results, and assisted in the writing. In Paper IV, I took part in the plan- ning and was responsible for the experiments. I also took part of the evalua- tion and the writing of the Paper as well as the algorithm implementation. In Paper V, I took part of the data evaluation as well as in the writing of Paper and the algorithm implementation. Contents 1. Introduction ...........................................................................................9 2. Liquid Separations...............................................................................11 2.1 Capillary Electrophoresis ...........................................................11 2.2 Liquid Chromatography .............................................................14 3. Mass Spectrometry ..............................................................................16 3.1 Electrospray Ionization...............................................................17 3.2 Mass Analyzers and Detectors ...................................................18 4 Hyphenated LS-MS and complex samples..........................................20 4.1 Applications................................................................................21 4.1.1 ‘The biomarker study’ ...........................................................21 4.1.2 ‘The paracetamol study’ ........................................................21 4.1.3 ‘The digest study’ ..................................................................22 4.1.4 ‘The beverage study’ .............................................................22 4.1.5 The use of CE vs. CEC with mass spectrometry ...................22 4.1.6 The use of CE vs. LC with mass spectrometry......................23 4.1.7 The use of TOF/Quadrupole/FTICR mass spectrometry.......23 5. Managing LS-MS data.........................................................................24 5.1 Software......................................................................................24 5.2 Generating data...........................................................................27 5.3 Preprocessing of spectra.............................................................27 5.4 Exporting data ............................................................................27 5.4 Applications................................................................................32 5.4.1 ‘The biomarker study’ ...........................................................32 5.4.2 ‘The paracetamol study’ ........................................................32 5.4.3 ‘The digest study’ ..................................................................33 5.4.4 ‘The beverage study’ .............................................................33 6. Exploring LS-MS Data........................................................................34 6.1 Exploring single runs..................................................................34 6.2 Comparing two runs ...................................................................35 6.2.1 Intensity scaling and normalization .......................................36 6.2.2 Peak alignment.......................................................................37 6.2.3 Data compression...................................................................40 6.3 Exploring the whole data set ......................................................41 6.3.1 The ‘fuzzy’ correlation matrix...............................................42 6.3.2 Cluster analysis and dendrograms .........................................42 6.3.3 PCA and scores......................................................................43 6.3.4 PCA and loadings ..................................................................45 6.3.5 Selection of scores by ANOVA.............................................46 6.3.6 Variable assessment with target correlation (contrasts).........47 7. Conclusions and future aspects............................................................50 8. Acknowledgements..............................................................................51 9. Summary in Swedish ...........................................................................52 9.1 Introduktion till LS/MS ..............................................................52 9.2 Hantering och utvärdering av LS/MS-data.................................53 9.4 Projekt ........................................................................................54 10. References.......................................................................................56 Abbreviations LS Liquid separation BPC Base peak chromatogram CE Capillary electrophoresis CEC Capillary electrochromatography COW Correlation optimized warping EOF Electroosmotic flow ESI Electrospray ionization FTICR Fourier transform ion cyclotron resonance i.d. Inner diameter LC Liquid chromatography MAD Median absolute deviation MALDI Matrix assisted laser desorption ionization MS Mass spectrometry PCA Principal component analysis SVD Singular value decomposition TIC Total ion chromatogram TOF Time-of-flight mass spectrometer XIC Extracted ion chromatogram 1. Introduction Analytical chemists need to analyze a broad range of complex samples, often with very low concentrations of the analytes in relation to the surrounding material (the matrix). The complexity of the samples as well as the wanted precision and accuracy influence the choice of the method(s) used. Sample pretreatment and separation is also crucial for generation of reliable and usable data. In view of this, an analytical chemist must be able to handle a range of de- tection methods, and realize how to best combine them with prior steps in the analytical chain. More recently, the handling of data has become more of an issue. With the techniques available today, it is easy to generate data cor- responding to a full DVD (4.7 GB), per analyst and hour. To handle data of that magnitude, we need computational power and lots of electronic memory (RAM) as well as storage space. A number of methods to extract the relevant information have been developed and ever more elaborate ones are still be- ing published at a high pace. This thesis is partly focusing on the manage- ment of data. It is possible today to generate a lot more data than the com- puters can handle without help and planning. The help and planning includes cunning containers for the data, i.e. file formats, and equally
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