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• W2912003126 abstract "Apolipoproteins are major structural and functional constituents of lipoprotein particles. As modulators of lipid metabolism, adipose tissue biology, and energy homeostasis, apolipoproteins may serve as biomarkers or potential therapeutic targets for cardiometabolic diseases. Mice are the preferred model to study metabolic disease and CVD, but a comprehensive method to quantify circulating apolipoproteins in mice is lacking. We developed and validated a targeted proteomics assay to quantify eight apolipoproteins in mice via proteotypic signature peptides and corresponding stable isotope-labeled analogs. The LC/MS/MS method requires only a 3 µl sample volume to simultaneously determine mouse apoA-I, apoA-II, apoA-IV, apoB-100, total apoB, apoC-I, apoE, and apoJ concentrations. ApoB-48 concentrations can be calculated by subtracting apoB-100 from total apoB. After we established the analytic performance (sensitivity, linearity, and imprecision) and compared results for selected apolipoproteins against immunoassays, we applied the method to profile apolipoprotein levels in plasma and isolated HDL from normocholesterolemic C57BL/6 mice and from hypercholesterolemic Ldl-receptor- and Apoe-deficient mice. In conclusion, we present a robust, quantitative LC/MS/MS method for the multiplexed analysis of eight apolipoproteins in mice. This assay can be applied to investigate the effects of genetic manipulation or dietary interventions on apolipoprotein levels in plasma and isolated lipoprotein fractions. Apolipoproteins are major structural and functional constituents of lipoprotein particles. As modulators of lipid metabolism, adipose tissue biology, and energy homeostasis, apolipoproteins may serve as biomarkers or potential therapeutic targets for cardiometabolic diseases. Mice are the preferred model to study metabolic disease and CVD, but a comprehensive method to quantify circulating apolipoproteins in mice is lacking. We developed and validated a targeted proteomics assay to quantify eight apolipoproteins in mice via proteotypic signature peptides and corresponding stable isotope-labeled analogs. The LC/MS/MS method requires only a 3 µl sample volume to simultaneously determine mouse apoA-I, apoA-II, apoA-IV, apoB-100, total apoB, apoC-I, apoE, and apoJ concentrations. ApoB-48 concentrations can be calculated by subtracting apoB-100 from total apoB. After we established the analytic performance (sensitivity, linearity, and imprecision) and compared results for selected apolipoproteins against immunoassays, we applied the method to profile apolipoprotein levels in plasma and isolated HDL from normocholesterolemic C57BL/6 mice and from hypercholesterolemic Ldl-receptor- and Apoe-deficient mice. In conclusion, we present a robust, quantitative LC/MS/MS method for the multiplexed analysis of eight apolipoproteins in mice. This assay can be applied to investigate the effects of genetic manipulation or dietary interventions on apolipoprotein levels in plasma and isolated lipoprotein fractions. Alterations of plasma lipoproteins, especially elevated levels of apoB-containing lipoproteins, have been causally related to the risk of atherosclerotic CVD (ASCVD) (1Weber C. Noels H. Atherosclerosis: current pathogenesis and therapeutic options.Nat. Med. 2011; 17: 1410-1422Crossref PubMed Scopus (1551) Google Scholar). Apolipoproteins are major structural and functional components of lipoproteins with diverse biologic functions. They can serve as cofactors for enzymes and ligands for cell-surface receptors (2Alaupovic P. Apolipoprotein composition as the basis for classifying plasma lipoproteins. Characterization of ApoA- and ApoB-containing lipoprotein families.Prog. Lipid Res. 1991; 30: 105-138Crossref PubMed Scopus (89) Google Scholar). Thereby, apolipoproteins are intimately involved in the metabolism of plasma cholesterol and triglycerides carried by lipoproteins. Blood concentrations of apoB and apoA-I, as surrogate measures for VLDL/LDL and HDL, have been proposed as superior markers for the assessment of cardiovascular risk in several trials (3McQueen M.J. Hawken S. Wang X. Ounpuu S. Sniderman A. Probstfield J. Steyn K. Sanderson J.E. Hasani M. Volkova E. INTERHEART study investigators Lipids, lipoproteins, and apolipoproteins as risk markers of myocardial infarction in 52 countries (the INTERHEART study): a case-control study.Lancet. 2008; 372: 224-233Abstract Full Text Full Text PDF PubMed Scopus (636) Google Scholar, 4Pencina M.J. D'Agostino R.B. Zdrojewski T. Williams K. Thanassoulis G. Furberg C.D. Peterson E.D. Vasan R.S. Sniderman A.D. Apolipoprotein B improves risk assessment of future coronary heart disease in the Framingham Heart Study beyond LDL-C and non-HDL-C.Eur. J. Prev. Cardiol. 2015; 22: 1321-1327Crossref PubMed Scopus (88) Google Scholar). In addition, apolipoproteins like apoA-II, apoA-IV, apoC-I, apoC-II, apoC-III, and apoE that can associate with various lipoprotein classes are also key players in lipid metabolism and may represent targets for the diagnosis and treatment of dyslipidemias and ASCVD (5Remaley A.T. Apolipoprotein A-II: still second fiddle in high-density lipoprotein metabolism?.Arterioscler. Thromb. Vasc. Biol. 2013; 33: 166-167Crossref PubMed Scopus (15) Google Scholar, 6Wang F. Fei W. Kohan A. Chun-Min L. Min L. Howles P. Tso P. Apolipoprotein A-IV: a protein intimately involved in metabolism.J. Lipid Res. 2015; 56: 1403-1418Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 7Mahley R.W. Apolipoprotein E: from cardiovascular disease to neurodegenerative disorders.J. Mol. Med. (Berl.). 2016; 94: 739-746Crossref PubMed Scopus (255) Google Scholar). In this context, Pechlaner et al. (8Pechlaner R. Tsimikas S. Yin X. Willeit P. Baig F. Santer P. Oberhollenzer F. Egger G. Witztum J.L. Alexander V.J. et al.Very-low-density lipoprotein-associated apolipoproteins predict cardiovascular events and are lowered by inhibition of APOC-III.J. Am. Coll. Cardiol. 2017; 69: 789-800Crossref PubMed Scopus (122) Google Scholar) recently reported associations of several apolipoproteins (including apoC-II, apoC-III, apoE, apoH, and apoL1) with incident CVD in a prospective population-based study, which raises the potential prospects of apolipoprotein profiling for CVD. Moreover, several apolipoproteins have been associated with metabolic functions in glucose homeostasis, insulin sensitivity, and adipose tissue biology and may thus contribute to obesity and diabetes mellitus (9Hoofnagle A.N. Wu M. Gosmanova A.K. Becker J.O. Wijsman E.M. Brunzell J.D. Kahn S.E. Knopp R.H. Lyons T.J. Heinecke J.W. Low clusterin levels in high-density lipoprotein associate with insulin resistance, obesity, and dyslipoproteinemia.Arterioscler. Thromb. Vasc. Biol. 2010; 30: 2528-2534Crossref PubMed Scopus (69) Google Scholar, 10Wang F. Kohan A.B. Kindel T.L. Corbin K.L. Nunemaker C.S. Obici S. Woods S.C. Davidson W.S. Tso P. Apolipo­protein A-IV improves glucose homeostasis by enhancing insulin secretion.Proc. Natl. Acad. Sci. USA. 2012; 109: 9641-9646Crossref PubMed Scopus (87) Google Scholar, 11Wassef H. Salem H. Bissonnette S. Baass A. Dufour R. Davignon J. Faraj M. White adipose tissue apolipoprotein C–I secretion in relation to delayed plasma clearance of dietary fat in humans.Arterioscler. Thromb. Vasc. Biol. 2012; 32: 2785-2793Crossref PubMed Scopus (17) Google Scholar, 12Zvintzou E. Lhomme M. Chasapi S. Filou S. Theodoropoulos V. Xapapadaki E. Kontush A. Spyroulias G. Tellis C.C. Tselepis A.D. et al.Pleiotropic effects of apolipoprotein C3 on HDL functionality and adipose tissue metabolic activity.J. Lipid Res. 2017; 58: 1869-1883Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). It is also established that apolipoproteins, such as apoJ and, in particular, apoE, contribute to the pathogenesis of Alzheimer's disease via multiple mechanisms (13Liao F. Yoon H. Kim J. Apolipoprotein E metabolism and functions in brain and its role in Alzheimer's disease.Curr. Opin. Lipidol. 2017; 28: 60-67PubMed Google Scholar, 14Zandl-Lang M. Fanaee-Danesh E. Sun Y. Albrecher N.M. Gali C.C. Čančar I. Kober A. Tam-Amersdorfer C. Stracke A. Storck S.M. et al.Regulatory effects of simvastatin and apoJ on APP processing and amyloid-β clearance in blood-brain barrier endothelial cells.Biochim. Biophys. Acta Mol. Cell. Biol. Lipids. 2018; 1863: 40-60Crossref PubMed Scopus (41) Google Scholar, 15Dong H.K. Gim J-A. Yeo S.H. Kim H-S. Integrated late onset Alzheimer's disease (LOAD) susceptibility genes: cholesterol metabolism and trafficking perspectives.Gene. 2017; 597: 10-16Crossref PubMed Scopus (33) Google Scholar) and that apolipoproteins can exhibit antiinflammatory and antioxidative functions (16Filou S. Lhomme M. Karavia E.A. Kalogeropoulou C. Theodoropoulos V. Zvintzou E. Sakellaropoulos G.C. Petropoulou P-I. Constantinou C. Kontush A. et al.Distinct roles of apolipoproteins A1 and E in the modulation of high-density lipoprotein composition and function.Biochemistry. 2016; 55: 3752-3762Crossref PubMed Scopus (41) Google Scholar, 17Murphy A.J. Hoang A. Aprico A. Sviridov D. Chin-Dusting J. Anti-inflammatory functions of apolipoprotein A-I and high-density lipoprotein are preserved in trimeric apolipoprotein A-I.J. Pharmacol. Exp. Ther. 2013; 344: 41-49Crossref PubMed Scopus (22) Google Scholar, 18Van Lenten B.J. Wagner A.C. Nayak D.P. Hama S. Navab M. Fogelman A.M. High-density lipoprotein loses its anti-inflammatory properties during acute influenza A infection.Circulation. 2001; 103: 2283-2288Crossref PubMed Scopus (301) Google Scholar). Importantly, current knowledge on apolipoprotein physiology and their specific roles in various diseases is still incomplete, which underlines the importance to further study specific apolipoproteins in experimental settings. The mouse is the most common experimental model to investigate metabolic disorders and CVD (19von Scheidt M. Zhao Y. Kurt Z. Pan C. Zeng L. Yang X. Schunkert H. Lusis A.J. Applications and limitations of mouse models for understanding human atherosclerosis.Cell Metab. 2017; 25: 248-261Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). At present, a comprehensive quantitative analysis of circulating apolipoproteins in mouse models is limited by a lack of suitable assays. Common methods to measure apolipoproteins in mice are Ab-based immunoassays, which suffer from typical drawbacks, such as lack of specific Abs, cross-reactivity, and high lot-to-lot variability (20Hoofnagle A.N. Wener M.H. The fundamental flaws of immunoassays and potential solutions using tandem mass spectrometry.J. Immunol. Methods. 2009; 347: 3-11Crossref PubMed Scopus (381) Google Scholar). Other limitations are the restriction to single-analyte testing and requirements of high sample volumes. On the contrary, the coupling of liquid chromatography and tandem mass spectrometry (LC/MS/MS) facilitates simultaneous quantitation of multiple proteins from low volumes of plasma with high throughput (20Hoofnagle A.N. Wener M.H. The fundamental flaws of immunoassays and potential solutions using tandem mass spectrometry.J. Immunol. Methods. 2009; 347: 3-11Crossref PubMed Scopus (381) Google Scholar, 21Cravatt B.F. Simon G.M. Yates J.R. The biological impact of mass-spectrometry-based proteomics.Nature. 2007; 450: 991-1000Crossref PubMed Scopus (571) Google Scholar, 22Hoofnagle A.N. Heinecke J.W. Lipoproteomics: using mass spectrometry-based proteomics to explore the assembly, structure, and function of lipoproteins.J. Lipid Res. 2009; 50: 1967-1975Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). To this end, surrogate proteotypic peptides, which are unique to the investigated proteins, are generated by enzymatic digestion, followed by LC separation and MS detection. In these targeted proteomics assays, stable isotope-labeled (SIL) peptides or proteins are used as internal standards (ISs), allowing the quantification of target proteins (23Shuford C.M. Walters J.J. Holland P.M. Sreenivasan U. Askari N. Ray K. Grant R.P. Absolute protein quantification by mass spectrometry: not as simple as advertised.Anal. Chem. 2017; 89: 7406-7415Crossref PubMed Scopus (48) Google Scholar). Work from our laboratory and others has previously demonstrated that LC/MS/MS assays are suitable for multiplexed analysis of middle- to high-abundant apolipoproteins in human plasma (8Pechlaner R. Tsimikas S. Yin X. Willeit P. Baig F. Santer P. Oberhollenzer F. Egger G. Witztum J.L. Alexander V.J. et al.Very-low-density lipoprotein-associated apolipoproteins predict cardiovascular events and are lowered by inhibition of APOC-III.J. Am. Coll. Cardiol. 2017; 69: 789-800Crossref PubMed Scopus (122) Google Scholar, 24Ceglarek U. Dittrich J. Becker S. Baumann F. Kortz L. Thiery J. Quantification of seven apolipoproteins in human plasma by proteotypic peptides using fast LC-MS/MS.Proteomics Clin. Appl. 2013; 7: 794-801Crossref PubMed Scopus (32) Google Scholar, 25Toth C.A. Kuklenyik Z. Jones J.I. Parks B.A. Gardner M.S. Schieltz D.M. Rees J.C. Andrews M.L. McWilliams L.G. Pirkle J.L. et al.On-column trypsin digestion coupled with LC-MS/MS for quantification of apolipoproteins.J. Proteomics. 2017; 150: 258-267Crossref PubMed Scopus (39) Google Scholar, 26van den Broek I. Romijn F.P.H.T.M. Nouta J. Van Der Laarse A. Drijfhout J.W. Smit N.P.M. van der Burgt Y.E.M. Cobbaert C.M. Automated multiplex LC-MS/MS assay for quantifying serum apolipoproteins A-I, B, C–I, C–II, C–III, and E with qualitative apolipoprotein E phenotyping.Clin. Chem. 2016; 62: 188-197Crossref PubMed Scopus (72) Google Scholar). However, a corresponding method to determine apolipoprotein levels in plasma of mice has not been described so far. In the present study, we developed and validated a targeted proteomics assay for the simultaneous quantification of eight apolipoproteins from only 3 µl of mouse plasma. We then applied this method to study plasma apolipoprotein concentrations in WT C57BL6/J mice and two different hypercholesterolemic mouse models, the apoE-deficient (ApoE0) and the LDL receptor-deficient (LDLR0) mouse, in fasted as well as fed state. Ammonium bicarbonate, iodoacetamide, 2,2,2-trifluoroethanol, and Tris(2-carboxyethyl)phosphine were acquired from Sigma-Aldrich (St. Louis, MO). Formic acid and trifluoroacetic acid were obtained from Fluka (Buchs, Switzerland). Murine recombinant apoA-I was purchased from Sino Biological (Beijing, China). Ultra-high-performance LC/MS grade methanol, acetonitrile, and 2-propanol were obtained from Biosolve (Valkenswaard, The Netherlands). Sequencing-grade modified trypsin was acquired from Promega (Madison, WI; catalog no. V5111). Ultrapure water from a Barnstead NANOpure water purification system (Thermo Fisher Scientific, Waltham, MA) was used. Male normocholesterolemic C57BL/6J mice (JAX strain no. 000664), hyperlipidemic ApoE0 (JAX strain no. 002052), and LDLR0 (JAX strain no. 002207) mice were kept on a regular chow diet and studied at 13 weeks of age. Blood samples were collected in EDTA tubes at 9 AM (random fed) and after an overnight fast in the same mice. Blood samples were centrifuged at 3,000 g for 10 min to obtain plasma, which was then used for the assay, processed to prepare lipoprotein fractions, or directly stored at −80°C until analysis. Lipoprotein fractions were isolated from 60 µl freshly prepared mouse plasma by sequential ultracentrifugation, as described previously (27Burkhardt R. Sündermann S. Ludwig D. Ceglarek U. Holdt L.M. Thiery J. Teupser D. Cosegregation of aortic root atherosclerosis and intermediate lipid phenotypes on chromosomes 2 and 8 in an intercross of C57BL/6 and BALBc/ByJ low-density lipoprotein receptor−/− mice.Arterioscler. Thromb. Vasc. Biol. 2011; 31: 775-784Crossref PubMed Scopus (10) Google Scholar). Subsequently, obtained lipoprotein fractions were stored at −80°C until analysis. Cholesterol and triglyceride levels were determined enzymatically using colorimetric assay kits (Roche Diagnostics). Protein concentration of isolated HDL was quantified using the Pierce BCA Kit (Thermo Fisher). All animal procedures were performed in accordance with the rules for animal care of the local government authorities and were approved by the animal care and use committee of Leipzig University as well as by the animal care committee of the Bezirksregierung Leipzig, Germany. Proteotypic peptides were selected according to accepted selection rules for each of the eight apolipoproteins (28Mohammed Y. Domański D. Jackson A.M. Smith D.S. Deelder A.M. Palmblad M. Borchers C.H. PeptidePicker: a scientific workflow with web interface for selecting appropriate peptides for targeted proteomics experiments.J. Proteomics. 2014; 106: 151-161Crossref PubMed Scopus (95) Google Scholar, 29Lange V. Picotti P. Domon B. Aebersold R. Selected reaction monitoring for quantitative proteomics: a tutorial.Mol. Syst. Biol. 2008; 4: 222Crossref PubMed Scopus (1121) Google Scholar). Peptide sequences containing cysteine or methionine residues as well as known polymorphisms and sites of posttranslational modifications were excluded. To ensure peptide specificity and to check for potential sequence overlaps, Blast searches against the NCBI database (http://blast.ncbi.nlm.nih.gov/Blast.cgi) were performed. Furthermore, proteotypic peptides were verified in MASCOT MS/MS ion searches (Matrix Science) against the SwissProt database after enhanced product ion analysis of tryptically digested plasma from LDLR0 mice. Peptides and SIL analogs thereof were synthesized by the Core Unit for Peptide Technology of the Interdisciplinary Center for Clinical Research (Faculty of Medicine, University of Leipzig). Synthesized peptides were purified to >98% using preparative reversed-phase HPLC. Purity and identity of the purified peptides were then evaluated by use of analytical HPLC and MALDI-MS. Peptide sequences are provided in supplemental Table S1. The individual peptides were stored as lyophilized aliquots at −80°C until use. At time of use, the lyophilized peptides were precisely weighed out and dissolved in 2-propanol/water (1:1, vol/vol) to prepare stock solutions at concentrations between 1 and 10 mmol/l. Peptide working standards (concentrated between 0.1 and 1 mmol/l) were then obtained by dilution of stock solutions with 100 mmol/l ammonium bicarbonate (see supplemental Table S2 for details). All peptide solutions were stored at −80°C. Storage, handling, and reconstitution of peptides followed the usage recommendations for MS-based assays (30Hoofnagle A.N. Whiteaker J.R. Carr S.A. Kuhn E. Liu T. Massoni S.A. Thomas S.N. Townsend R.R. Zimmerman L.J. Boja E. et al.Recommendations for the generation, quantification, storage, and handling of peptides used for mass spectrometry-based assays.Clin. Chem. 2016; 62: 48-69Crossref PubMed Scopus (146) Google Scholar). With each batch of samples, an in-house nine point peptide calibration series was carried along, which was equivalently processed as the study samples. The highest-concentrated calibrators were produced from peptide working standards. Lower-concentrated calibrators were then prepared by serial dilution thereof with 100 mmol/l ammonium bicarbonate. Calibration curves prepared in 100 mmol/l ammonium bicarbonate buffer were also tested against calibration curves prepared in plasma to establish the parallelism of the response in buffer and in plasma matrix for the calibrators (supplemental Fig. S1). The workflow and all concentrations of the single calibrators for each apolipoprotein are summarized in supplemental Table S2. Calibration curves were plotted using analyte-to-IS peak area ratios. Linear regression was accomplished applying 1/× weighting. Data processing was performed with Multiquant 2.0 (Sciex). EDTA plasma, lipoprotein fractions, and calibrators were treated according to a previously established standardized sample-preparation protocol (24Ceglarek U. Dittrich J. Becker S. Baumann F. Kortz L. Thiery J. Quantification of seven apolipoproteins in human plasma by proteotypic peptides using fast LC-MS/MS.Proteomics Clin. Appl. 2013; 7: 794-801Crossref PubMed Scopus (32) Google Scholar). In brief, 3 µl of study sample or calibrator were diluted 1:2 with a SIL peptide mix prepared in 100 mmol/l ammonium bicarbonate. Final IS concentrations are summarized in supplemental Table S1. Denaturation was performed using 6.9 mol/l 2,2,2-trifluoroethanol. For reduction of disulfide bonds, samples were incubated with 5 mmol/l Tris(2-carboxyethyl)phosphine for 30 min at 60°C. Subsequently, alkylation was performed for 30 min with 10 mmol/l iodoacetamide at room temperature in the dark. After 1:10 dilution with 100 mmol/l ammonium bicarbonate, 8 µg of trypsin dissolved in 50 mmol/l acetic acid was added. Tryptic digestion was performed for 16 h at 37°C. Digestion kinetics had been investigated using a plasma pool of C57BL/6J mice (n = 5), which had been incubated with trypsin for 0.5, 1, 2, 4, 8, 16, 20, and 24 h (supplemental Fig. S2). Digestion was stopped with 0.1% formic acid. Sample clean-up was performed by solid-phase extraction using 10 mg of Oasis HLB 1cc Flangless Vac Cartridges (Waters, Milford, MA) followed by drying under nitrogen. Afterward, samples were reconstituted in 1 ml of 0.1% formic acid in methanol/water (eluent A; 10/90, vol/vol) and were centrifuged for 5 min at 14,500 g. Supernatants were used for LC/MS/MS analysis. LC/MS/MS analysis was adapted from a previously published method for the analysis of human apolipoproteins developed in our laboratory (24Ceglarek U. Dittrich J. Becker S. Baumann F. Kortz L. Thiery J. Quantification of seven apolipoproteins in human plasma by proteotypic peptides using fast LC-MS/MS.Proteomics Clin. Appl. 2013; 7: 794-801Crossref PubMed Scopus (32) Google Scholar). The microLC equipment included an Ultimate® 3000 TCC-3000SD Column Oven, an Ultimate® 3000 RS Autosampler, and an Ultimate® 3000 RSLCnano System (Thermo Scientific Dionex, Sunnyvale, CA). A QTRAP® 5500 (Sciex, Darmstadt, Germany) equipped with a Turbo V™ ion spray source and controlled by Analyst 1.5.1 software was used for MS detection in multiple reaction monitoring (MRM) mode. Chromatographic conditions were controlled by CHROMELEON 6.80 software (Thermo Scientific Dionex). A ZORBAX 300SB-C18 column (150 × 1.0 mm inner diameter, 3.5 µm particle size) combined with its corresponding guard column (Agilent Technologies, Santa Clara, CA) was used for chromatographic separation at 40°C. The autosampler temperature was set to 10°C, and the injection volume was 1 µl. Binary gradient elution was performed within a total run time of 7.6 min using eluent A and 0.1% formic acid in methanol/water (90/10, vol/vol) as eluent B. The LC time program was adjusted as follows: linear increase from 20% B to 100% B in 3.5 min, 100% B until 6.5 min, and reequilibration from 6.6 to 7.6 min with 20% B. The flow rate was set to 50 µl/min. MRM transitions are summarized in supplemental Table S1. Two mass transitions were monitored per peptide. Positive ESI was performed applying the following settings: ion spray voltage = 5,500 V, ion source heater temperature = 400°C, source gas 1 = 20 psi, source gas 2 = 50 psi, and curtain gas = 35 psi. Q1 and Q3 were used in unit resolution. To assess assay precision, pooled plasma samples from C57BL/6J, LDLR0, and ApoE0 mice (n = 4–6 per strain) were used. The plasma pools were analyzed for at least five times on 1 day or for a single time on at least five consecutive working days to evaluate within- and between-day imprecision, respectively. Concentrations quantified at signal-to-noise ratios of 3 or 10 were defined as LOD or lower limit of quantification (LLOQ), respectively. To assess the influence of sample storage conditions, apolipoproteins were quantified in plasma samples stored at 4°C within 24 h or after 4 days; in plasma samples stored at −80°C for 2 years or in samples that underwent multiple freeze-thaw cycles (n = 3) over 6 weeks before sample preparation. Recovery of apoA-I was assessed by spiking murine recombinant apoA-I into pooled plasma samples of C57BL/6J mice, resulting in two different concentration levels. Furthermore, the results obtained for apoE and apoB-100 from the LC/MS/MS assay were compared with commercially available immunoassays (Mabtech, 3752-1HP-2; Abcam, ab230932), which were performed according to the manufacturers' recommendations. For method-comparison purposes, samples with intermediate apolipoprotein concentrations were prepared by mixing plasma pools of hyperlipidemic mice (LDLR0 and ApoE0) with pooled plasma of WT mice at different ratios. Data are presented as mean ± SD unless otherwise noted. Results were analyzed by Student's t-test (two-tailed) or by ANOVA with Dunnett's posttest, when multiple comparisons were made. The influence of sample storage conditions and freeze-thaw cycles were evaluated using acceptable change limits criteria according to ISO 5725-6 (31Oddoze C. Lombard E. Portugal H. Stability study of 81 analytes in human whole blood, in serum and in plasma.Clin. Biochem. 2012; 45: 464-469Crossref PubMed Scopus (199) Google Scholar, 32International Organization for Standardization.Accuracy (trueness and precision) of measurement methods and results–part 6: use in practice of accuracy values. 1994; Google Scholar). Method comparison with immunoassays was performed by Passing-Bablok regression and Bland-Altmann plot analysis. Data were analyzed using GraphPad Prism software (GraphPad Software, San Diego, CA) or MedCalc software (MedCalc Software, Ostend, Belgium) (for Passing-Bablok and Bland-Altman). Statistical significance was defined as P < 0.05. We developed a high-throughput LC/MS/MS assay to simultaneously quantify apoA-I, apoA-II, apoA-IV, apoB-total, apoB-100, apoC-I, apoE, and apoJ in mouse plasma via proteotypic signature peptides and corresponding SIL analogs. Monitored peptides and respective mass transitions are presented in supplemental Table S1. The sample-preparation procedure was optimized for reproducible quantitation of murine apolipoproteins from only 3 µl of sample. ISs were added at the very beginning of the sample processing to compensate potential sample losses. The complete sample- preparation workflow is summarized in Fig. 1. Digestion kinetics of the eight investigated apolipoproteins, which are summarized in supplemental Fig. S2, confirmed that all selected signature peptides were released within 16 h of tryptic digestion. Furthermore, proteotypic peptides and SIL analogs thereof were stable during trypsin incubation. As VLDL secreted from mouse liver contains both apoB-48 (consisting of the N-terminal 48% of apoB-100) and full-length apoB-100, we established separate assays for apoB-100 and total apoB, which further allows the calculation of apoB-48 plasma levels. For this reason, we analyzed peptide INIDIPLPLGGK [amino acids (aa) 1344-1355], which is present in apoB-48 as well as in apoB-100 and therefore determines total plasma apoB. In addition, the apoB-100-specific peptide DLDVVNIPLAR (aa 2,739–2,749) was monitored. Hence, apoB-48 concentrations were calculated by subtracting apoB-100 from total apoB concentrations. Sensitivity and linearity of the developed LC/MS/MS assay were established in plasma samples of normocholesterolemic chow-fed C57/BL6 mice. Determined LODs were between 0.03 µmol/l for apoA-IV, apoB-100, and apoJ and 0.2 µmol/l for apoC-I (Table 1). LLOQs ranged from 0.1 µmol/l for apoJ to 0.7 µmol/l for apoC-I (Table 1). The lower limits of the quantification range were defined by the assay's LLOQ (for apoB-100) or the expected apolipoprotein concentrations in WT mice. The upper limits of the quantification range were adapted to allow the reliable analysis of increased apolipoprotein concentrations in hypercholesterolemic mice (Table 1).TABLE 1LODs, LLOQs, and quantification ranges of analyzed apolipoproteinsProteinLODLLOQQuantification Rangeµmol/lapoA-I0.070.231.3–240apoA-II0.160.531.3–240apoA-IV0.030.110.2–15apoB-total0.120.390.6–15apoB-1000.030.110.1–6apoC-I0.200.670.7–27apoE0.080.280.7–129apoJ0.030.100.1–4.8 Open table in a new tab The assay's within-day and between-day imprecision was first determined by use of a plasma pool from chow-fed C57BL/6 mice. As shown in Table 2, within-day imprecision was found between 1.2% (apoB-total) and 10.9% (apoJ), whereas between-day imprecision ranged from 4.5% (apoA-II) to 11.2% (apoC-I), with the exception of apoB-100 (31.1%). Assay precision was also evaluated in plasma pools from LDLR0 and APOE0 mice, demonstrating similar coefficients of variation in hyperlipidemic samples (see supplemental Table S3). Of note, between-day imprecision for apoB-100 was only 5.3% in LDLR0 mice, which are characterized by elevated apoB-100. Hence, the high coefficient of variation (CV) for apoB-100 in C57/BL6 mice likely resulted from the very low apoB-100 plasma concentrations at the LLOQ.TABLE 2Within-day and between-day imprecision of the assayWithin-Day Imprecision (n = 10)Between-Day Imprecision (n = 8)ProteinMean, µmol/lCV, %Mean, µmol/lCV, %apoA-I46.075.147.686.7apoA-II60.727.057.174.5apoA-IV3.376.13.206.4apoB-total2.321.22.218.0apoB-1000.228.50.1331.1apoC-I9.158.49.0911.2apoE2.777.12.807.4apoJ1.9810.91.916.2 Open table in a new tab We further exemplarily evaluated the recovery of the assay by spiking pool plasma samples from C57BL/6 mice with recombinant murine apoA-I at two different concentrations. As shown in Table 3, respective recovery rates of apoA-I in murine plasma were 101.3% and 101.9%.TABLE 3Recovery of recombinant apoA-IProteinInitial Conc.Added Conc.Expected Conc.Measured Conc.Recovery Rateµmol/l%apoA-I48206869.3 (± 6.6)101.9apoA-I48105858.8 (± 1." @default.
• W2912003126 created "2019-02-21" @default.
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• W2912003126 date "2019-04-01" @default.
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• W2912003126 title "Simultaneous LC/MS/MS quantification of eight apolipoproteins in normal and hypercholesterolemic mouse plasma" @default.
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