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Lipoproteomics II: Mapping of proteins in high-density lipoprotein using two-dimensional gel electrophoresis and mass spectrometry
Linköping University, Department of Clinical and Experimental Medicine, Occupational and Environmental Medicine . Linköping University, Faculty of Health Sciences.
Linköping University, Department of Clinical and Experimental Medicine, Occupational and Environmental Medicine . Linköping University, Faculty of Health Sciences.
Linköping University, Department of Clinical and Experimental Medicine, Occupational and Environmental Medicine . Linköping University, Faculty of Health Sciences.
Linköping University, Department of Clinical and Experimental Medicine, Occupational and Environmental Medicine . Linköping University, Faculty of Health Sciences.
2005 (English)In: Proteomics, ISSN 1615-9853, Vol. 5, no 5, 1431-1445 p.Article in journal (Refereed) Published
Abstract [en]

High-density lipoprotein (HDL) is the most abundant lipoprotein particle in the plasma and a negative risk factor of atherosclerosis. By using a proteomic approach it is possible to obtain detailed information about its protein content and protein modifications that may give new information about the physiological roles of HDL. In this study the two subfractions; HDL2 and HDL3, were isolated by two-step discontinuous density-gradient ultracentrifugation and the proteins were separated with two-dimensional gel electrophoresis and identified with peptide mass fingerprinting, using matrix-assisted laser desorption/ionisation time of flight mass spectrometry. Identified proteins in HDL were: the dominating apo A-I as six isoforms, four of them with a glycosylation pattern and one of them with retained propeptide, apolipoprotein (apo) A-II, apo A-IV, apo C-I, apo C-II, apo C-III (two isoforms), apo E (five isoforms), the recently discovered apo M (two isoforms), serum amyloid A (two isoforms) and serum amyloid A-IV (six isoforms). Furthermore, alpha-1-antitrypsin was identified in HDL for the first time. Additionally, salivary alpha-amylase was identified as two isoforms in HDL2, and apo L and a glycosylated apo A-II were identified in HDL3. Besides confirming the presence of different apolipoproteins, this study indicates new patterns of glycosylated apo A-I and apo A-II. Furthermore, the study reveals new proteins in HDL; alpha-1-antitrypsin and salivary alpha-amylase. Further investigations about these proteins may give new insight into the functional role of HDL in coronary artery diseases.

Place, publisher, year, edition, pages
2005. Vol. 5, no 5, 1431-1445 p.
Keyword [en]
High-density lipoprotein, Matrix-assisted laser desorption/ionisation-time of flight-mass spectrometry, Two-dimensional gel electrophoresis
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-14351DOI: 10.1002/pmic.200401010OAI: oai:DiVA.org:liu-14351DiVA: diva2:23299
Available from: 2007-03-15 Created: 2007-03-15 Last updated: 2013-09-20
In thesis
1. Lipoproteomics: A New Approach to the Identification and Characterization of Proteins in LDL and HDL
Open this publication in new window or tab >>Lipoproteomics: A New Approach to the Identification and Characterization of Proteins in LDL and HDL
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

A proteomic approach was applied to examine the protein composition of low-density lipoprotein (LDL) and high-density lipoprotein (HDL) in humans. LDL and HDL were isolated by density gradient ultracentrifugation, and proteins were separated with twodimensional gel electrophoresis (2-DE) and identified with peptide mass fingerprinting, using matrix-assisted laser desorption/ionization-time of flight mass spectrometry, and with amino acid sequencing using electrospray ionization tandem mass spectrometry. To improve the identification of low abundant proteins in silver stained 2-DE gels, 2,5-dihydroxybenzoic acid was used instead of α-cyano-4-hydroxycinnamic acid as matrix in the peptide mass fingerprinting procedure; this was demonstrated to give more matching peptide peaks, higher sequence coverage, and higher signal to noise ratio. Altogether 18 different proteins were demonstrated in LDL and/or HDL: three of these (calgranulin A, lysozyme C and transthyretin) have not been identified in LDL before. Apo C-II, apo C-III, apo E, apo A-I, apo A-IV, apo J, apo M, serum amyloid A-IV and α1-antitrypsin were found in both LDL and HDL, while apo B-100 (clone), calgranulin A, lysozyme C and transthyretin were found only in LDL, and apo A-II, apo C-I, and serum amyloid A only in HDL. Salivary α-amylase wass identified only in HDL2, and apo L and glycosylated apo A-II only in HDL3. Many of the proteins occurred in a number of isoforms: in all, 47 different isoform identities were demonstrated. A 2-DE mobility shift assay and deglycosylation experiments were used to demonstrate, for the first time, that apo M in LDL and HDL occurs in five isoforms; three that are both N-glycosylated and sialylated, one that is N-glycosylated but not sialylated and one that is neither N-glycosylated nor sialylated. LDL from obese subjects was found to contain more apo J, apo C-II, apo M, α1-antitrypsin and serum amyloid A-IV than LDL from controls,, and also more of an acidic isoform (pI/Mr; 5.2 / 23 100) of apo A-I. In addition, the new LDLassociated protein transthyretin, was found to be significantly more abundant in LDL from obese subjects. On the other hand, the amounts of apo A-IV and the major isoform of apo A-I (pI/Mr; 5.3 / 23 100) were significantly less. Altogether, these findings (i) illustrate the power of 2-DE and mass spectrometry for detailed mapping of the proteins and their isoforms in human lipoproteins; (ii) demonstrate the presence of a number of new proteins in LDL (calgranulin A, lysozyme C and transthyretin); (iii) give precise biochemical clues to the polymorphism of apo M in LDL and HDL, and; (iv) indicate that obesity is associated with significant changes in the protein profile of LDL. It is concluded that new information on lipoproteins can easily be obtained through a proteomic approach, thus facilitating the development of a new proteomic field: lipoproteomics. Much further investigation in this field is warranted, particularly because newly discovered LDL and HDL proteins may play hitherto unknown role(s) in inflammatory reactions of the arterial wall and evolve as useful biomarkers in cardiovascular disease.

Place, publisher, year, edition, pages
Institutionen för molekylär och klinisk medicin, 2007
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 986
Keyword
LDL, HDL, Proteomik, Atheroskleros, Masspektrometri, 2-DE
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:liu:diva-8527 (URN)978-91-85715-47-3 (ISBN)
Public defence
2007-03-29, Aulan, Hälsans Hus, Campus US, Linköpings Universitet, Linköping, 09:00 (English)
Opponent
Supervisors
Available from: 2007-03-15 Created: 2007-03-15 Last updated: 2009-08-22
2. Lipoproteomics: mapping of proteins in LDL and HDL
Open this publication in new window or tab >>Lipoproteomics: mapping of proteins in LDL and HDL
2005 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

High-density lipoprotein (HDL) and (LDL)-density lipoprotein (LDL) are important lipoprotein fractions in human plasma. HDL is the most abundant lipoprotein particle and a negative risk factor of atherosclerosis while LDL is considered to possess atherogenic properties. The molecular mechanisms underlying the relationship between LDU/HDL and the development of atherosclerosis are not clear. Therefore, detailed information about the protein composition of LDU/HDL may contribute to reveal their role in atherogenesis and the mechanisms that lead to or protect from coronary disease in humans. Here, we sought to map the proteins in human LDL/HDL by a proteomic approach.

LDL and HDL were isolated by two-step discontinuous density-gradient ultracentrifugation and the proteins were separated with two-dimensional gel electrophoresis and identified with peptide mass fingerprinting, using matrix assisted laser desorption/ionization-time of flight-mass spectrometry and with amino acid sequencing using electrospray ionization tandem mass spectrometry.

In LDL these procedures identified apo B-100, apo C-II, apo C-I (three isoforms), apo E (four isoforms), apo A-I (three isoforms), apo A-IV, apo J and apo M (three isoforms not previously described). In addition, three proteins that have not previously been identified in LDL were found: serum amyloid A-IV (two isoforms). calgranulin A and lysozyme C. The identities of apo M, calgranulin A, and lysozyme C were confirmed by sequence information obtained after collision-induced dissociation fragmentation of pep tides characteristic for these proteins. Moreover, the presence of lysozyme C was further corroborated by demonstrating enriched hydrolytic activity in LDL against Micrococcus lysodeikticus.

Identified proteins in HDL were: the dominating apo A-1 as seven isoforms, four of them with a modification pattern and one of them with retained propeptide, apo A-II, apo A-IV, apo C-I. apo C-II, apo C-III (two isoforms), apo E (five isoforms), apo J, the recently discovered apo M (two isoforms), serum amyloid A (two isoforms) and serum amyloid A-IV (six isoforms). Furthermore, alpha-1-antitrypsin was identified in HDL for the first time. Additionally, salivary alpha-amylase was identified as two isoforms in HDL2 , and apo L and aglycosylatcd apo A-II were identified in HDL3.

These results indicate that both LDL and HDL contains a number of apolipoproteins, many of them occurs in different isoforms. The demonstration, for the first time, that LDL contains calgranulin A, lysozyme C and apo J raises the possibility that LDL proteins may play hitherto unknown role(s) in immune and inflammatory reactions of the arterial wall. Additionally, new patterns of glycosylated apo A-I and apo A-II and new proteins; alpha-1-antitrypsin and salivary alpha-amylase were detected in HDL. Further investigations about these proteins may give new insight into the functional role of LDL and HDL in coronary artery disease.

Publisher
40 p.
Series
Linköping Studies in Health Sciences. Thesis, ISSN 1100-6013 ; 73
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-31069 (URN)16791 (Local ID)91-8529-917-0 (ISBN)16791 (Archive number)16791 (OAI)
Available from: 2009-10-09 Created: 2009-10-09 Last updated: 2013-09-20

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Karlsson, HelenLeandersson, PerTagesson, ChristerLindahl, Mats

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