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| 2. |
- Kultima, Kim, et al.
(författare)
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Biomarkers of disease and post-mortem changes : Heat stabilization, a necessary tool for measurement of protein regulation
- 2011
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Ingår i: Journal of Proteomics. - : Elsevier BV. - 1874-3919 .- 1876-7737. ; 75:1, s. 145-159
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Forskningsöversikt (refereegranskat)abstract
- This review focuses on post sampling changes and how the Stabilizor system has been used to control this natural biological process and potential implications on cancer-specific biomarkers due to post sampling changes. Tissue sampling is a major traumatic event that can have drastic effects within a very short timeframe at the molecular level [1] resulting in loss of sample quality due to post-mortem changes. A heat-stabilization technology, using the Stabilizor system, has been developed to quickly and permanently abolish the enzymatic activity that causes these changes post-sampling and so preserve sample quality. The Stabilizor system has been shown to give better sample quality when analyzing a variety of tissues in various proteomic workflows. In this paper we discuss the impact of using heat-stabilized tissue in different proteomic applications. Based on our observations regarding the overlap between commonly changing proteins and proteins found to change post-mortem we also highlight a group of proteins of particular interest in cancer studies.
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| 3. |
- Pettersson, Camilla, 1978, et al.
(författare)
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LDL-associated apolipoprotein J and lysozyme are associated with atherogenic properties of LDL found in type 2 diabetes and the metabolic syndrome.
- 2011
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Ingår i: Journal of internal medicine. - : Wiley. - 1365-2796 .- 0954-6820. ; 269:3, s. 306-321
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Tidskriftsartikel (refereegranskat)abstract
- Objectives Exchangeable low-density lipoprotein (LDL)-associated proteins can affect the atherogenic properties of LDL. Our aim was to analyze the protein composition of LDL from individuals with or without type 2 diabetes and the metabolic syndrome (T2DM) in relation to other LDL-particle characteristics, to assess whether certain proteins associate more with certain subclasses of LDL typical for T2DM, such as small, apoCIII-rich LDL. Design LDL from two cohorts of 61-year-old men (n = 19 and 64) with or without T2DM was isolated using size-exclusion chromatography or deuterium oxide-based ultracentrifugation. LDL-associated proteins were identified using mass spectrometry and quantified using two-dimensional gel electrophoresis or enzyme-linked immunosorbent assay. Differently expressed LDL-associated proteins apolipoprotein (apo)J and lysozyme were also measured in serum from a third cohort of women (n = 71) with or without T2DM. Lysozyme binding to advanced glycation end product (AGE)-LDL was examined in vitro. Results ApoJ and lysozyme were increased in LDL particles with increased apoCIII content and decreased cholesterol content. When isolated with SEC, LDL from individuals with T2DM contained more apoJ and lysozyme and less apoA1 than LDL from control individuals. LDL content of apoJ correlated with a smaller LDL-particle size. Serum levels of lysozyme, but not apoJ, were increased in individuals with T2DM. In vitro, lysozyme associated more with AGE-LDL than with unmodified LDL. Conclusions Our results indicate that apoJ and lysozyme are increased in LDL with characteristics of small dense LDL in T2DM. Small dense LDL is easily glycated, and the increased affinity of lysozyme for AGE-LDL provides a possible partial explanation for an increase of lysozyme from those with type 2 diabetes.
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| 5. |
- Scholz, Birger, et al.
(författare)
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Impact of Temperature Dependent Sampling Procedures in Proteomics and Peptidomics : A Characterization of the Liver and Pancreas Post Mortem Degradome
- 2011
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Ingår i: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 10:3, s. M900229MCP200-
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Tidskriftsartikel (refereegranskat)abstract
- Little is known about the nature of post mortem degradation of proteins and peptides on a global level, the so-called degradome. This is especially true for nonneural tissues. Degradome properties in relation to sampling procedures on different tissues are of great importance for the studies of, for instance, post translational modifications and/or the establishment of clinical biobanks. Here, snap freezing of fresh(< 2 min post mortem time) mouse liver and pancreas tissue is compared with rapid heat stabilization with regard to effects on the proteome (using two-dimensional differential in-gel electrophoresis) and peptidome (using label free liquid chromatography). We report several proteins and peptides that exhibit heightened degradation sensitivity, for instance superoxide dismutase in liver, and peptidyl-prolyl cis-trans isomerase and insulin C-peptides in pancreas. Tissue sampling based on snap freezing produces a greater amount of degradation products and lower levels of endogenous peptides than rapid heat stabilization. We also demonstrate that solely snap freezing related degradation can be attenuated by subsequent heat stabilization. We conclude that tissue sampling involving a rapid heat stabilization step is preferable to freezing with regard to proteomic and peptidomic sample quality.
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- Stingl, Christoph, et al.
(författare)
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Uncovering Effects of Ex Vivo Protease Activity during Proteomics and Peptidomics Sample Extraction in Rat Brain Tissue by Oxygen-18 Labeling
- 2014
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 13:6, s. 2807-2817
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Tidskriftsartikel (refereegranskat)abstract
- In biological samples, proteins and peptides are altered by proteolytic activity. The actual ex vivo form of the peptidome or proteome analyzed, therefore, does not always reflect the natural in vivo state. Sample stabilization and sample treatment are thereby decisive for how far these two states diverge. To assess ex vivo formation of peptides, we used enzymatic incorporation of oxygen-18 water during proteolysis (PALeO approach) to label ex-vivo-formed peptides in rodent brain tissue. Rates of ex-vivo-formed peptides were determined in 25 samples that were stabilized and treated by six different protocols, whereby samples were subjected to different conditions such as temperature, urea concentration, and duration of treatment. Samples were measured by nano LC-Orbitrap-MS, and incorporation of oxygen-18 was determined by MS/MS database search and analysis of the precursor isotope pattern. Extent of ex vivo degradations was affected relevantly by the sample treatment protocol applied and stopped almost completely by heat stabilization. Determination of the formation state by oxygen-18 incorporation by MS/MS database search correlated well to more elaborate analysis of the MS isotope pattern. Overall, oxygen-18 labeling in combination with shotgun data-acquisition and MS/MS database search offers an adjuvant and easily applicable tool to monitor sample quality and fidelity in peptide and neuropeptide sample preparations.
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| 7. |
- Zadravec, Damir, 1980-, et al.
(författare)
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Ablation of the very-long-chain fatty acid elongase ELOVL3 in mice leads to constrained lipid storage and resistance to diet-induced obesity.
- 2010
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Ingår i: The FASEB journal : official publication of the Federation of American Societies for Experimental Biology. - : Wiley. - 1530-6860 .- 0892-6638. ; 24:11, s. 4366-77
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Tidskriftsartikel (refereegranskat)abstract
- Although saturated and monounsaturated very-long-chain fatty acids (VLCFAs) have long been associated with undesirable effects on health, including obesity, heart failure, and atherosclerosis, the physiological role of endogenous synthesis is largely unknown. The fatty acid elongase ELOVL3 is involved in the synthesis of C20-C24 saturated and monounsaturated VLCFAs mainly in liver, brown and white adipose tissue, and triglyceride-rich glands such as the sebaceous and meibomian glands. Here we show that ablation of ELOVL3 leads to reduced adiponectin levels, constrained expansion of adipose tissue, and resistance against diet-induced obesity, a situation that is more exaggerated in female mice. Both female and male knockout mice show reduced hepatic lipogenic gene expression and triglyceride content, a situation that is associated with reduced de novo fatty acid synthesis and uptake. As a consequence, the VLDL-triglyceride level in serum is significantly reduced. Remarkably, despite increased energy expenditure, markedly reduced serum levels of leptin, and increased expression of orexigenic peptides in the hypothalamus, the Elovl3(-/-) mice do not compensate by increased food intake. Thus, these results reveal that C20-C22 saturated and monounsaturated VLCFAs produced by ELOVL3 are indispensable for appropriate synthesis of liver triglycerides, fatty acid uptake, and storage in adipose tissue.
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