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Strategies to explore the membrane proteome of a cell
Stockholm University, Faculty of Science, Department of Analytical Chemistry.
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The cell envelope plays key roles in numerous processes such as maintaining cellular integrity, communication with other cells, signal transduction, maintenance of cellular homeostasis, and regulation of the traffic of molecules between the cell and the extracellular milieu. Essential membrane components in many of these processes are proteins. It is estimated that ~20-30% of the predicted open reading frames (ORFs) of all organisms encode membrane proteins. Furthermore, two thirds of drug targets are membrane proteins. However, despite their importance, membrane proteins have so far been mostly neglected in most proteomic studies, due to the inherent challenges in analyzing them.

The focus of this thesis is to devise strategies that allow investigation of membrane proteins and their associated complexes. Optimization of sample preparation in the underlying studies has allowed important goals to be reached in membrane protein analyses at various levels such as elucidation of their primary structure by collision-induced dissociation (CID) and electron-capture dissociation (ECD) mass spectrometry (MS), profiling membrane proteins and their complexes, the discovery of novel protein complexes, definition of their topology, and unambiguous identification of protein-bound ligand(s). This thesis paves the way for better characterization of membrane proteins and their assemblies hinting towards the crucial role(s) they play in maintaining normal cell physiology.

Place, publisher, year, edition, pages
Stockholm: Department of Analytical Chemistry, Stockholm University , 2011. , 94 p.
Keyword [en]
Membrane proteins, Proteomics, Escherichia coli, Enterococcus faecalis, Synechocystis, Mass Spectrometry, BN/SDS-PAGE, FT-ICR-MS, Myelin P2 protein
National Category
Analytical Chemistry
Research subject
Analytical Chemistry
Identifiers
URN: urn:nbn:se:su:diva-56848ISBN: 978-91-7447-211-0 (print)OAI: oai:DiVA.org:su-56848DiVA: diva2:413507
Public defence
2011-06-08, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript. Available from: 2011-05-12 Created: 2011-04-28 Last updated: 2012-01-24Bibliographically approved
List of papers
1. Proteomics of Synechocystis sp. PCC 6803 Identification of novel integral plasma membrane proteins: Identification of novel integral plasma membrane proteins
Open this publication in new window or tab >>Proteomics of Synechocystis sp. PCC 6803 Identification of novel integral plasma membrane proteins: Identification of novel integral plasma membrane proteins
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2007 (English)In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 274, no 3, 791-804 p.Article in journal (Refereed) Published
Abstract [en]

The cyanobacterial plasma membrane is an essential cell barrier with functions such as the control of taxis, nutrient uptake and secretion. These functions are carried out by integral membrane proteins, which are difficult to identify using standard proteomic methods. In this study, integral proteins were enriched from purified plasma membranes of Synechocystis sp. PCC 6803 using urea wash followed by protein resolution in 1D SDS/PAGE. In total, 51 proteins were identified by peptide mass fingerprinting using MALDI-TOF MS. More than half of the proteins were predicted to be integral with 1–12 transmembrane helices. The majority of the proteins had not been identified previously, and include members of metalloproteases, chemotaxis proteins, secretion proteins, as well as type 2 NAD(P)H dehydrogenase and glycosyltransferase. The obtained results serve as a useful reference for further investigations of the address codes for targeting of integral membrane proteins in cyanobacteria.

Keyword
cyanobacteria, integral proteins, plasma membrane, proteome, Synechocystis 6803
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:su:diva-56160 (URN)10.1111/j.1742-4658.2006.05624.x (DOI)
Available from: 2011-04-11 Created: 2011-04-11 Last updated: 2017-12-11Bibliographically approved
2. Systematic Analysis of Native Membrane Protein Complexes in Escherichia coli
Open this publication in new window or tab >>Systematic Analysis of Native Membrane Protein Complexes in Escherichia coli
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2011 (English)In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 10, no 4, 1848-1859 p.Article in journal (Refereed) Published
Abstract [en]

The cell envelope of Escherichia coli is an essential structure that modulates exchanges between the cell and the extra-cellular milieu. Previous proteomic analyses have suggested that it contains a significant number of proteins with no annotated function. To gain insight into these proteins and the general organization of the cell envelope proteome, we have carried out a systematic analysis of native membrane protein complexes. We have identified 30 membrane protein complexes (6 of which are novel) and present reference maps that can be used for cell envelope profiling. In one instance, we identified a protein with no annotated function (YfgM) in a complex with a well-characterized periplasmic chaperone (PpiD). Using the guilt by association principle, we suggest that YfgM is also part of the periplasmic chaperone network. The approach we present circumvents the need for engineering of tags and protein overexpression. It is applicable for the analysis of membrane protein complexes in any organism and will be particularly useful for less-characterized organisms where conventional strategies that require protein engineering (i.e., 2-hybrid based approaches and TAP-tagging) are not feasible.

Keyword
Escherichia coli, cell envelope, proteome, membrane protein, protein complex, BN-PAGE, PpiD, YfgM
National Category
Analytical Chemistry Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-56162 (URN)10.1021/pr101105c (DOI)000288924000038 ()21210718 (PubMedID)
Available from: 2011-04-11 Created: 2011-04-11 Last updated: 2017-12-11Bibliographically approved
3. A reference map of the membrane proteome of Enterococcus faecalis
Open this publication in new window or tab >>A reference map of the membrane proteome of Enterococcus faecalis
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2011 (English)In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 11, no Special issue, 3935-3941 p.Article in journal (Refereed) Published
Abstract [en]

Enterococcus faecalis is a gram-positive bacterium that is part of the indigenous microbiotica of humans and animals as well as an opportunistic pathogen. In this study, we have fractionated the membrane proteome of E. faecalis and identified many of its constituents by mass spectrometry. We present blue native-/SDS-PAGE reference maps that contain 102 proteins. These proteins are important for cellular homeostasis, virulence, and antibiotic intervention. Intriguingly, many proteins with no known function were also identified, indicating that there are substantial gaps in the knowledge of this organism's biology. On a more limited scale, we also provide insight into the composition of membrane protein complexes. This study is a first step toward elucidating the membrane proteome of E. faecalis, which is critical for a better understanding of how this bacterium interacts with a host and with the extracellular milieu.

Keyword
Blue native-PAGE; Enterococcus faecalis; Membrane proteome; Microbiology; Nano-LC-ESI-MS/MS; Protein complex
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:su:diva-65507 (URN)10.1002/pmic.201100103 (DOI)000296142100019 ()
Available from: 2011-12-12 Created: 2011-12-12 Last updated: 2017-12-08Bibliographically approved
4. Porcine P2 myelin protein primary structure and bound fatty acids determined by mass spectrometry
Open this publication in new window or tab >>Porcine P2 myelin protein primary structure and bound fatty acids determined by mass spectrometry
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2010 (English)In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 397, no 5, 1903-1910 p.Article in journal (Refereed) Published
Abstract [en]

Complementary collision-induced/electron capture dissociation Fourier-transform ion cyclotron resonance mass spectrometry was used to fully sequence the protein P2 myelin basic protein. It is an antigenic fatty-acid-binding protein that can induce experimental autoimmune neuritis: an animal model of Guillain-Barre syndrome, a disorder similar in etiology to multiple sclerosis. Neither the primary structure of the porcine variant, nor the fatty acids bound by the protein have been well established to date. A 1.8-angstrom crystal structure shows but a bound ligand could not be unequivocally identified. A protocol for ligand extraction from protein crystals has been developed with subsequent gas chromatography MS analysis allowing determination that oleic, stearic, and palmitic fatty acids are associated with the protein. The results provide unique and general evidence of the utility of mass spectrometry for characterizing proteins from natural sources and generating biochemical information that may facilitate attempts to elucidate the causes for disorders such as demyelination.

Keyword
CID, ECD, De novo sequencing, GC/MS, Lipid identification, Myelin proteins
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:su:diva-50527 (URN)10.1007/s00216-010-3762-0 (DOI)000278810000032 ()
Note
authorCount :8Available from: 2010-12-28 Created: 2010-12-28 Last updated: 2017-12-11Bibliographically approved

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