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Proteomics and metabolomics in biological and medical applications
Umeå University, Faculty of Science and Technology, Department of Chemistry.
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Biological processes in living organisms consist of a vast number of different molecular networks and interactions, which are complex and often hidden from our understanding. This work is focused on recovery of such details for two quite distant examples: acclimation to extreme freezing tolerance in Siberian spruce (Picea obovata) and detection of proteins associated with prostate cancer.

The first biological system in the study, upon P. obovata, is interesting by this species ability to adapt and sustain extremely low temperatures, such as -60⁰C or below. Despite decades of investigations, the essential features and mechanisms of the amazing ability of this species still remains unclear. To enhance knowledge about extreme freezing tolerance, the metabolome and proteome of P. obovata’s needles were collected during the tree’s acclimation period, ranging from mid August to January, and have been analyzed.

The second system within this study is the plasma proteome analysis of high risk prostate cancer (PCa) patients, with and without bone metastases. PCa is one of the most common cancers among Swedish men, which can abruptly develop into an aggressive, lethal disease. The diagnostic tools, including PSA-tests, are insufficient in predicting the disease’s aggressiveness and novel prognostic markers are urgently required.

Both biological systems have been analyzed following similar steps: by two-dimensional difference gel electrophoresis (2D-DIGE) techniques, followed by protein identification using mass spectrometry (MS) analysis and multivariate methods. Data processing has been utilized for searching for proteins that serve as unique indicators for characterizing the status of the systems. In addition, the gas chromatography-mass spectrometry (GC-MS) study of the metabolic content of P.obovata’s needles, from the extended observation period, has been performed. The studies of both systems, combined with thorough statistical analysis of experimental outcomes, have resulted in novel insights and features for both P. obovata and prostate cancer. In particular, it has been shown that dehydrins, Hsp70s, AAA+ ATPases, lipocalin and several proteins involved in cellular metabolism etc., can be uniquely associated with acclimation to extreme freezing in conifers. Metabolomic analysis of P. obovata needles has revealed systematic metabolic changes in carbohydrate and lipid metabolism. Substantial increase of raffinose, accumulation of desaturated fatty acids, sugar acids, sugar alcohols, amino acids and polyamines that may act as compatible solutes or cryoprotectants have all been observed during the acclimation process.

Relevant proteins for prostate cancer progression and aggressiveness have been identified in the plasma proteome study, for patients with and without bone metastasis. Proteins associated with lipid transport, coagulation, inflammation and immune response have been found among them. 

Place, publisher, year, edition, pages
Umeå: Kemiska institutionen, Umeå Universitet , 2011. , 44 p.
Keyword [en]
2D-DIGE, biomarkers, cold-acclimation, conifer, freezing tolerance, GC-MS, metabolomics, multiple hypothesis test, multivariate analysis, OPLS-DA, Picea obovata, plasma, prostate cancer, proteomics, ProteoMiner, Siberian spruce
National Category
Chemical Sciences
Identifiers
URN: urn:nbn:se:umu:diva-43520ISBN: 978-91-7459-204-7OAI: oai:DiVA.org:umu-43520DiVA: diva2:414446
Public defence
2011-05-25, KBC-huset, KB3A9, Umeå Universitet, Umeå, 10:00 (English)
Opponent
Supervisors
Available from: 2011-05-04 Created: 2011-05-03 Last updated: 2014-12-10Bibliographically approved
List of papers
1. Proteomics of extreme freezing tolerance in Siberian spruce (Picea obovata)
Open this publication in new window or tab >>Proteomics of extreme freezing tolerance in Siberian spruce (Picea obovata)
2010 (English)In: Journal of Proteomics, ISSN 1874-3919, Vol. 73, no 5, 965-975 p.Article in journal (Refereed) Published
Abstract [en]

Differential expression of proteins in needles of the extreme freeze tolerant conifer Picea obovata during September, October and November was analyzed using DIGE technology and multivariate analysis. More than 1200 spots were detected, and the abundance of 252 of these spots was significantly altered during the course of acclimation. The 252 spots were clustered into five distinct expression profiles. Among the protein spots showing differential expression, 43 were identified by MALDI-TOF/TOF and twelve of them matched proteins associated with various biotic and abiotic stress responses in other plants. Dehydrins, Hsp70s, AAA+ ATPases, lipocalin, cyclophilins, glycine-rich protein (GNP) and several reactive oxygen intermediate scavenging proteins showed increased accumulation levels from September to November. The expression profiles and putative role of the identified proteins during acclimation and freezing tolerance are discussed.

Place, publisher, year, edition, pages
Elsevier, 2010
Keyword
freezing tolerance, Picea obovata, DIGE, MALDI-TOF/TOF, OPLS-DA, dehydrins
Identifiers
urn:nbn:se:umu:diva-43519 (URN)10.1016/j.jprot.2009.12.010 (DOI)000276167900010 ()
Available from: 2011-05-03 Created: 2011-05-03 Last updated: 2011-05-05Bibliographically approved
2. Metabolomic analysis of extreme freezing tolerance in Siberian spruce (Picea obovata)
Open this publication in new window or tab >>Metabolomic analysis of extreme freezing tolerance in Siberian spruce (Picea obovata)
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2014 (English)In: New Phytologist, ISSN 0028-646X, E-ISSN 1469-8137, Vol. 204, no 3, 545-555 p.Article in journal (Refereed) Published
Abstract [en]

Siberian spruce (Picea obovata) is one of several boreal conifer species that can survive at extremely low temperatures (ELTs). When fully acclimated, its tissues can survive immersion in liquid nitrogen. Relatively little is known about the biochemical and biophysical strategies of ELT survival. We profiled needle metabolites using gas chromatography coupled with mass spectrometry (GC-MS) to explore the metabolic changes that occur during cold acclimation caused by natural temperature fluctuations. In total, 223 metabolites accumulated and 52 were depleted in fully acclimated needles compared with pre-acclimation needles. The metabolite profiles were found to develop in four distinct phases, which are referred to as pre-acclimation, early acclimation, late acclimation and fully acclimated. Metabolite changes associated with carbohydrate and lipid metabolism were observed, including changes associated with increased raffinose family oligosaccharide synthesis and accumulation, accumulation of sugar acids and sugar alcohols, desaturation of fatty acids, and accumulation of digalactosylglycerol. We also observed the accumulation of protein and nonprotein amino acids and polyamines that may act as compatible solutes or cryoprotectants. These results provide new insight into the mechanisms of freezing tolerance development at the metabolite level and highlight their importance in rapid acclimation to ELT in P.obovata.

Keyword
acclimation, cold, conifer, frost, GC-MS, metabolites, needles, Siberian spruce (Picea obovata)
National Category
Botany
Identifiers
urn:nbn:se:umu:diva-96810 (URN)10.1111/nph.12950 (DOI)000343869000014 ()
Note

Originally included in thesis in manuscript form.

Available from: 2014-12-10 Created: 2014-12-03 Last updated: 2014-12-10Bibliographically approved
3. Pair-wise multicomparison and OPLS analyses of cold-acclimation phases in Siberian spruce
Open this publication in new window or tab >>Pair-wise multicomparison and OPLS analyses of cold-acclimation phases in Siberian spruce
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2012 (English)In: Metabolomics, ISSN 1573-3882, Vol. 8, no Suppl 1, 123-130 p.Article in journal (Refereed) Published
Abstract [en]

Analysis of metabolomics data often goes beyond the task of discovering biomarkers and can be aimed at recovering other important characteristics of observed metabolomic changes. In this paper we explore different methods to detect the presence of distinctive phases in seasonal-responsive changes of metabolomic patterns of Siberian spruce (Picea obovata) during cold acclimation occurred in the period from mid-August to January. Multivariate analysis, specifically orthogonal projection to latent structures discriminant analysis (OPLSDA), identified time points where the metabolomic patterns underwent substantial modifications as a whole, revealing four distinctive phases during acclimation. This conclusion was re-examined by a univariate analysis consisting of multiple pair-wise comparisons to identify homogeneity intervals for each metabolite. These tests complemented OPLS-DA, clarifying biological interpretation of the classification: about 60% of metabolites found responsive to the cold stress indeed changed at one or more of the time points predicted by OPLS-DA. However, the univariate approach did not support the proposed division of the acclimation period into four phases: less than 10% of metabolites altered during the acclimation had homogeneous levels predicted by OPLS-DA. This demonstrates that coupling the classification found by OPLS-DA and the analysis of dynamics of individual metabolites obtained by pair-wise multicomparisons reveals a more correct characterization of biochemical processes in freezing tolerant trees and leads to interpretations that cannot be deduced by either method alone. The combined analysis can be used in other ‘omics’-studies, where response factors have a causal dependence (like the time in the present work) and pairwise multicomparisons are not conservative.

Place, publisher, year, edition, pages
Springer, 2012
Keyword
metabolomics, multiple hypothesis test, multivariate analysis, OPLS-DA, Siberian spruce, cold-acclimation
National Category
Chemical Sciences
Identifiers
urn:nbn:se:umu:diva-43509 (URN)10.1007/s11306-011-0304-5 (DOI)
Note
Published online 11 april 2011. Copywright, The Author(s) 2011. This article is published with open access at Springerlink.comAvailable from: 2011-05-03 Created: 2011-05-03 Last updated: 2012-08-09Bibliographically approved
4. Two-dimensional difference gel electrophoresis to reveal proteins in plasma associated with high risk prostate cancer
Open this publication in new window or tab >>Two-dimensional difference gel electrophoresis to reveal proteins in plasma associated with high risk prostate cancer
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Prostate cancer is a common but highly variable disease. Conventional methods for prognostication are limited and needed to be complemented by novel biomarkers which could identify clinically significant tumors at a curable timepoint. With the aim to find biomarkers in plasma for high risk prostate cancer, we combined the ProteoMiner technology for protein fractionation with 2-dimentional difference gel electrophoresis (2D-DIGE). Plasma samples from patients with high risk tumors, defined to have bone metastases (M1, N=7) or locally advanced or poorly differentiated prostate cancer (M0, N=14), or benign disease (N=15) were analyzed. As a result of combined univariate and multivariate analyses (orthogonal partial least-squares discriminant analysis, OPLS-DA), 338 protein spots were found to be significantly associated with high risk prostate cancer. Ninety-eight (98) of the spots were successfully identified by LC-MS/MS, and OPLS-DA of those resulted in a reliable method for class separation; M1 vs. M0 vs. B (R2Xcum: 31.1%, R2Y cum: 59.9%, Q2cum: 41.4%, P < 0.0001). The panel of identified potential protein markers for high risk prostate cancer included highly to intermediately abundant plasma proteins involved in key processes such as lipid transport, coagulation, inflammation, and immune responses. Their putative roles for prostate cancer progression are discussed.

Keyword
biomarkers, 2D-DIGE, plasma, prostate cancer, proteomics, PorteoMiner
Identifiers
urn:nbn:se:umu:diva-43511 (URN)
Available from: 2011-05-03 Created: 2011-05-03 Last updated: 2011-05-04Bibliographically approved

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