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1.
PLoS One ; 13(4): e0194797, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29634782

RESUMO

Lipoproteins are complex molecular assemblies that are key participants in the intricate cascade of extracellular lipid metabolism with important consequences in the formation of atherosclerotic lesions and the development of cardiovascular disease. Multiplexed mass spectrometry (MS) techniques have substantially improved the ability to characterize the composition of lipoproteins. However, these advanced MS techniques are limited by traditional pre-analytical fractionation techniques that compromise the structural integrity of lipoprotein particles during separation from serum or plasma. In this work, we applied a highly effective and gentle hydrodynamic size based fractionation technique, asymmetric flow field-flow fractionation (AF4), and integrated it into a comprehensive tandem mass spectrometry based workflow that was used for the measurement of apolipoproteins (apos A-I, A-II, A-IV, B, C-I, C-II, C-III and E), free cholesterol (FC), cholesterol esters (CE), triglycerides (TG), and phospholipids (PL) (phosphatidylcholine (PC), sphingomyelin (SM), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and lysophosphatidylcholine (LPC)). Hydrodynamic size in each of 40 size fractions separated by AF4 was measured by dynamic light scattering. Measuring all major lipids and apolipoproteins in each size fraction and in the whole serum, using total of 0.1 ml, allowed the volumetric calculation of lipoprotein particle numbers and expression of composition in molar analyte per particle number ratios. Measurements in 110 serum samples showed substantive differences between size fractions of HDL and LDL. Lipoprotein composition within size fractions was expressed in molar ratios of analytes (A-I/A-II, C-II/C-I, C-II/C-III. E/C-III, FC/PL, SM/PL, PE/PL, and PI/PL), showing differences in sample categories with combinations of normal and high levels of Total-C and/or Total-TG. The agreement with previous studies indirectly validates the AF4-LC-MS/MS approach and demonstrates the potential of this workflow for characterization of lipoprotein composition in clinical studies using small volumes of archived frozen samples.


Assuntos
Apolipoproteínas/sangue , Cromatografia Líquida/métodos , Fracionamento por Campo e Fluxo/métodos , Lipídeos/sangue , Lipoproteínas/sangue , Espectrometria de Massas em Tandem/métodos , Apolipoproteína A-I/metabolismo , Apolipoproteína B-100/metabolismo , Análise Química do Sangue/métodos , Calibragem , Colesterol/química , Humanos , Luz , Modelos Estatísticos , Tamanho da Partícula , Controle de Qualidade , Espalhamento de Radiação , Fluxo de Trabalho
2.
Proteomics Clin Appl ; 11(7-8)2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28296203

RESUMO

PURPOSE: Apolipoprotein A-I (ApoA-I) and apolipoprotein B-100 (ApoB-100) are amphipathic proteins that are strong predictors of cardiovascular disease risk. The traceable calibration of apolipoprotein assays is a persistent challenge, especially for ApoB-100, which cannot be solubilized in purified form. EXPERIMENTAL DESIGN: A simultaneous quantitation method for ApoA-I and ApoB-100 was developed using tryptic digestion without predigestion reduction and alkylation, followed by LC separation coupled with isotope dilution MS analysis. The accuracy of the method was assured by selecting structurally exposed signature peptides, optimal choice of detergent, protein:enzyme ratio, and incubation time. Peptide calibrators were value assigned by isobaric tagging isotope dilution MS amino acid analysis. RESULTS: The method reproducibility was validated in technical repeats of three serum samples, giving 2-3% intraday CVs (N = 5) and <7% interday CVs (N = 21). The repeated analysis of interlaboratory harmonization standards showed -1% difference for ApoA-I and -12% for ApoB-100 relative to the assigned value. The applicability of the method was demonstrated by repeated analysis of 24 patient samples with a wide range of total cholesterol and triglyceride levels. CONCLUSIONS AND CLINICAL RELEVANCE: The method is applicable for simultaneous analysis of ApoA-I and ApoB-100 in patient samples, and for characterization of serum pool calibrators for other analytical platforms.


Assuntos
Apolipoproteína A-I/química , Apolipoproteína B-100/química , Análise Química do Sangue/métodos , Espectrometria de Massas , Fragmentos de Peptídeos/sangue , Proteólise , Tripsina/metabolismo , Sequência de Aminoácidos , Apolipoproteína A-I/metabolismo , Apolipoproteína B-100/metabolismo , Calibragem , Cromatografia Líquida , Humanos , Isótopos/química , Modelos Lineares , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Reprodutibilidade dos Testes
3.
J Proteomics ; 150: 258-267, 2017 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-27667389

RESUMO

Apolipoproteins measured in plasma or serum are potential biomarkers for assessing metabolic irregularities that are associated with the development of cardiovascular disease (CVD). LC-MS/MS allows quantitative measurement of multiple apolipoproteins in the same sample run. However, the accuracy and precision of the LC-MS/MS measurement depends on the reproducibility of the enzymatic protein digestion step. With the application of an immobilized enzyme reactor (IMER), the reproducibility of the trypsin digestion can be controlled with high precision via flow rate, column volume and temperature. In this report, we demonstrate the application of an integrated IMER-LC-MS/MS platform for the simultaneous quantitative analysis of eight apolipoproteins. Using a dilution series of a characterized serum pool as calibrator, the method was validated by repeated analysis of pooled sera and individual serum samples with a wide range of lipid profiles, all showing intra-assay CV<4.4% and inter-assay CV<8%. In addition, the method was compared with traditional homogeneous digestion coupled LC-MS/MS for the quantification of apoA-I and apoB-100. Applied in large scale human population studies, this method can serve the translation of a wider panel of apolipoprotein biomarkers from research to clinical application. SIGNIFICANCE: Currently, the translation of apolipoprotein biomarkers to clinical application is impaired because of the high cost of large cohort studies using traditional single-analyte immunoassays. The application of on-line tryptic digestion coupled with LC-MS/MS analysis is an effective way to address this problem. In this work we demonstrate a high throughput, multiplexed, automated proteomics workflow for the simultaneous analysis of multiple proteins.


Assuntos
Apolipoproteínas/análise , Proteólise , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Tripsina/metabolismo , Apolipoproteínas/metabolismo , Biomarcadores/sangue , Análise Química do Sangue/métodos , Proteínas Sanguíneas/análise , Proteínas Sanguíneas/metabolismo , Cromatografia Líquida , Estudos de Viabilidade , Humanos , Reprodutibilidade dos Testes
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