Preparative Electrophoresis for HDL Particle Size Separation and Intact-Mass Apolipoprotein Proteoform Analysis
The most abundant proteins on high-density lipoproteins (HDLs), apolipoproteins A-I (APOA1) and A-II (APOA2), are determinants of HDL function with 15 and 9 proteoforms (chemical-structure variants), respectively. The relative abundance of these proteoforms in human serum is associated with HDL cholesterol efflux capacity, and cholesterol content. However, the association between proteoform concentrations and HDL size is unknown. We employed a novel native-gel electrophoresis technique, clear native gel-eluted liquid fraction entrapment electrophoresis (CN-GELFrEE) paired with mass spectrometry of intact proteins to investigate this association. Pooled serum was fractionated using acrylamide gels of lengths 8 and 25 cm. Western blotting determined molecular diameter and intact-mass spectrometry determined proteoform profiles of each fraction. The 8- and 25 cm experiments generated 19 and 36 differently sized HDL fractions, respectively. The proteoform distribution varied across size. Fatty-acylated APOA1 proteoforms were associated with larger HDL sizes (Pearson's R = 0.94, p = 4 × 10-7) and were approximately four times more abundant in particles larger than 9.6 nm than in total serum; HDL-unbound APOA1 was acylation-free and contained the pro-peptide proAPOA1. APOA2 proteoform abundance was similar across HDL sizes. Our results establish CN-GELFrEE as an effective lipid-particle separation technique and suggest that acylated proteoforms of APOA1 are associated with larger HDL particles.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2023 |
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| Erschienen: |
2023 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:22 |
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| Enthalten in: |
Journal of proteome research - 22(2023), 5 vom: 05. Mai, Seite 1455-1465 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Lloyd-Jones, Cameron [VerfasserIn] |
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| Links: |
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| Anmerkungen: |
Date Completed 08.05.2023 Date Revised 30.11.2023 published: Print-Electronic Citation Status MEDLINE |
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| doi: |
10.1021/acs.jproteome.2c00804 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| PPN (Katalog-ID): |
NLM355578344 |
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| 500 | |a Citation Status MEDLINE | ||
| 520 | |a The most abundant proteins on high-density lipoproteins (HDLs), apolipoproteins A-I (APOA1) and A-II (APOA2), are determinants of HDL function with 15 and 9 proteoforms (chemical-structure variants), respectively. The relative abundance of these proteoforms in human serum is associated with HDL cholesterol efflux capacity, and cholesterol content. However, the association between proteoform concentrations and HDL size is unknown. We employed a novel native-gel electrophoresis technique, clear native gel-eluted liquid fraction entrapment electrophoresis (CN-GELFrEE) paired with mass spectrometry of intact proteins to investigate this association. Pooled serum was fractionated using acrylamide gels of lengths 8 and 25 cm. Western blotting determined molecular diameter and intact-mass spectrometry determined proteoform profiles of each fraction. The 8- and 25 cm experiments generated 19 and 36 differently sized HDL fractions, respectively. The proteoform distribution varied across size. Fatty-acylated APOA1 proteoforms were associated with larger HDL sizes (Pearson's R = 0.94, p = 4 × 10-7) and were approximately four times more abundant in particles larger than 9.6 nm than in total serum; HDL-unbound APOA1 was acylation-free and contained the pro-peptide proAPOA1. APOA2 proteoform abundance was similar across HDL sizes. Our results establish CN-GELFrEE as an effective lipid-particle separation technique and suggest that acylated proteoforms of APOA1 are associated with larger HDL particles | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Research Support, N.I.H., Extramural | |
| 650 | 4 | |a Research Support, Non-U.S. Gov't | |
| 650 | 4 | |a APOA1 | |
| 650 | 4 | |a HDL | |
| 650 | 4 | |a apolipoproteins | |
| 650 | 4 | |a cardiovascular disease | |
| 650 | 4 | |a cholesterol/efflux | |
| 650 | 4 | |a proteomics | |
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| 700 | 1 | |a Dos Santos Seckler, Henrique |e verfasserin |4 aut | |
| 700 | 1 | |a DiStefano, Nicholas |e verfasserin |4 aut | |
| 700 | 1 | |a Sniderman, Allan |e verfasserin |4 aut | |
| 700 | 1 | |a Compton, Phillip D |e verfasserin |4 aut | |
| 700 | 1 | |a Kelleher, Neil L |e verfasserin |4 aut | |
| 700 | 1 | |a Wilkins, John T |e verfasserin |4 aut | |
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| 856 | 4 | 0 | |u http://dx.doi.org/10.1021/acs.jproteome.2c00804 |3 Volltext |
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