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Evaluation of the SELDI-TOF MS technique for protein profiling of pancreatic islets exposed to glucose and oleate
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
2007 (English)In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 7, no 17, 3105-3115 p.Article in journal (Refereed) Published
Abstract [en]

The aim of the study was to evaluate the SELDI-TOF MS technique for pancreatic islet research. Mouse islets were cultured at low or high glucose levels in the absence or presence of oleate and characterized by measuring insulin secretion and oxygen tension. Subsequently, the islets were protein profiled. Up to 200 different peaks could be detected in a single experiment with the majority of peaks corresponding to proteins with masses below 30 kDa. By combining different protein arrays, the number of detected peaks could be increased further. The optimal binding of islet proteins was achieved using the anionic exchange array and phosphate buffer (pH 6) when the binding of insulin was low, which allowed other less abundant proteins to be captured. When islets from different culture conditions were profiled and analyzed, in total 25 proteins were found to be oleate/glucose-regulated. An oleate-regulated protein was chosen for identification work, which was conducted by passive elution from SDS-PAGE gels and subsequent in-gel trypsin digestion and MALDI-TOF MS. The protein was identified as peptidyl-prolyl isomerase B (PPI-B). In conclusion, the study demonstrates that SELDI-technique can be used not only to obtain islet protein patterns but is also helpful in the subsequent identification of differentially expressed proteins.

Place, publisher, year, edition, pages
2007. Vol. 7, no 17, 3105-3115 p.
Keyword [en]
Fatty acids, Islet, Peptidyl-prolyl isomerase B, Protein profiling, SELDI-TOF MS
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-96735DOI: 10.1002/pmic.200601019ISI: 000249635200008OAI: oai:DiVA.org:uu-96735DiVA: diva2:171410
Available from: 2008-02-21 Created: 2008-02-21 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Protein Profiling and Type 2 Diabetes
Open this publication in new window or tab >>Protein Profiling and Type 2 Diabetes
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Type 2 diabetes mellitus (T2DM) is a heterogeneous disease affecting millions of people worldwide. Both genetic and environmental factors contribute to the pathogenesis. The disease is characterized by alterations in many genes and their products. Historically, genomic alterations have mainly been studied at the transcriptional level in diabetes research. However, transcriptional changes do not always lead to altered translation, which makes it important to measure changes at the protein level. Proteomic techniques offer the possibility of measuring multiple protein alterations simultaneously.

In this thesis, the proteomic technique surface enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF MS) has been applied and evaluated in the context of T2DM research. Protocols for pancreatic islet and serum/plasma protein profiling and identification have been developed. In addition, the technique was used to analyze the influence of genetic background versus diabetic environment by determining serum protein profiles of individuals with normal glucose tolerance (NGT) and T2DM with or without family history of diabetes. In total thirteen serum proteins displayed different levels in serum from persons with NGT versus patients with T2DM. Among these proteins, apolipoprotein CIII, albumin and one yet unidentified protein could be classified as being changed because of different genetic backgrounds. On the other hand, ten proteins for instance transthyretin, differed as a result of the diabetic environment.

When plasma protein patterns of NGT and T2DM individuals characterized by differences in early insulin responses (EIR) were compared, nine proteins were found to be varying between the two groups. Of these proteins five were identified, namely two forms of transthyretin, hemoglobin α-chain, hemoglobin β-chain and apolipoprotein H. However no individual protein alone could explain the differences in EIR. In conclusion, SELDI-TOF MS has been successfully used in the context of T2DM research to identify proteins associated with family history of diabetes and β-bell function.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2008. 63 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 312
Keyword
Cell biology, Type 2 diabetes, Protein profiling, SELDI-TOF MS, Proteomics, Cellbiologi
Identifiers
urn:nbn:se:uu:diva-8458 (URN)978-91-554-7090-6 (ISBN)
Public defence
2008-03-14, Auditorium minus, Gustavianum, Akademigatan 3, Uppsala, 09:00
Opponent
Supervisors
Available from: 2008-02-21 Created: 2008-02-21Bibliographically approved

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