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Lipoproteomics: A New Approach to the Identification and Characterization of Proteins in LDL and HDL
Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Health Sciences.
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 [en]
LDL, HDL, Proteomik, Atheroskleros, Masspektrometri, 2-DE
National Category
Cell and Molecular Biology
Identifiers
URN: urn:nbn:se:liu:diva-8527ISBN: 978-91-85715-47-3 (print)OAI: oai:DiVA.org:liu-8527DiVA: diva2:23303
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
List of papers
1. Lipoproteomics I: Mapping of proteins in low-density lipoprotein using two-dimensional gel electrophoresis and mass spectrometry
Open this publication in new window or tab >>Lipoproteomics I: Mapping of proteins in low-density lipoprotein using two-dimensional gel electrophoresis and mass spectrometry
2005 (English)In: Proteomics, ISSN 1615-9853, Vol. 5, no 2, 551-565 p.Article in journal (Refereed) Published
Abstract [en]

The molecular mechanisms underlying the relationship between low-density lipoprotein (LDL) and the risk of atherosclerosis are not clear. Therefore, detailed information about the protein composition of LDL may contribute to reveal its role in atherogenesis and the mechanisms that lead to coronary disease in humans. Here, we sought to map the proteins in human LDL by a proteomic approach. LDL was 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. These procedures identified apo B-100, apo C-II, apo C-III (three isoforms), apo E (four isoforms), apo A-I (two 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 peptides characteristic for these proteins. Moreover, the presence of lysozyme C was further corroborated by demonstrating enriched hydrolytic activity in LDL against Micrococcus lysodeikticus. These results indicate that in addition to the dominating apo B-100, LDL contains a number of other apolipoproteins, many of which occur in different isoforms. The demonstration, for the first time, that LDL contains calgranulin A and lysozyme C raises the possibility that LDL proteins may play hitherto unknown role(s) in immune and inflammatory reactions of the arterial wall.

Keyword
Low-density lipoprotein, Lysozyme, Matrix assisted laser desorption/ionization-time of flight-mass spectrometry, Tandem mass spectrometry, Two-dimensional gel electrophoresis
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-14350 (URN)10.1002/pmic.200300938 (DOI)
Available from: 2007-03-15 Created: 2007-03-15 Last updated: 2013-09-20
2. Lipoproteomics II: Mapping of proteins in high-density lipoprotein using two-dimensional gel electrophoresis and mass spectrometry
Open this publication in new window or tab >>Lipoproteomics II: Mapping of proteins in high-density lipoprotein using two-dimensional gel electrophoresis and mass spectrometry
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.

Keyword
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:nbn:se:liu:diva-14351 (URN)10.1002/pmic.200401010 (DOI)
Available from: 2007-03-15 Created: 2007-03-15 Last updated: 2013-09-20
3. Peptide mass fingerprint data from silver stained proteins can be improved by using 2,5-dihydroxybenzoic acid instead of α-cyano-4-hydroxycinnamic acid as matrix in MALDI-TOF MS
Open this publication in new window or tab >>Peptide mass fingerprint data from silver stained proteins can be improved by using 2,5-dihydroxybenzoic acid instead of α-cyano-4-hydroxycinnamic acid as matrix in MALDI-TOF MS
Show others...
2007 (English)Article in journal (Refereed) Submitted
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-14352 (URN)
Available from: 2007-03-15 Created: 2007-03-15
4. Characterization of apolipoprotein M isoforms in low-density lipoprotein
Open this publication in new window or tab >>Characterization of apolipoprotein M isoforms in low-density lipoprotein
2006 (English)In: Journal of proteome research, ISSN 1535-3893, Vol. 5, no 10, 2685-2690 p.Article in journal (Refereed) Published
Abstract [en]

Apo M is a recently discovered human lipoprotein thought to be involved in the metabolism of lipids and lipoprotein particles. Here, a proteomic approach was applied to examine the glycosylation pattern of apo M in human LDL. We treated LDL proteins with N-glycosidase or neuraminidase, studied mobility shifts of Apo M by two-dimensional gel electrophoresis, and different isoforms were then identified with mass spectrometry. This way, we demonstrated the presence of five isoforms of apo M in LDL:  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. As judged from the examination of LDL from 20 healthy human subjects, the three N-glycosylated and sialylated forms are most abundant (80−100% of the total apo M in LDL) whereas the unsialylated and unglycosylated variants constitute at most 20%. Comparative analysis showed that the same five isoforms of apo M are also present in HDL. Further studies aiming at elucidating the role of apo M in health and disease will have to take this polymorphism of apo M proteins into account.

Keyword
apolipoprotein M, apo M, low-density lipoprotein, LDL, two-dimensional gel electrophoresis, 2-DE, MALDI-TOF mass spectrometry, glycosylations, sialylations
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-14353 (URN)10.1021/pr060180x (DOI)
Available from: 2007-03-15 Created: 2007-03-15
5. Comparative proteomics of lowdensity lipoprotein from normal weight and obese adults
Open this publication in new window or tab >>Comparative proteomics of lowdensity lipoprotein from normal weight and obese adults
Manuscript (Other academic)
Identifiers
urn:nbn:se:liu:diva-14354 (URN)
Available from: 2007-03-15 Created: 2007-03-15 Last updated: 2010-01-13

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