Lucia Renee Ruhaak (Presenter)
Leiden University Medical Center
Bio: Renee Ruhaak is a passionate analytical scientist with a strong interest in the application of novel technology to better understand changes in physiology associated with states of health and disease. She obtained her PhD from Leiden University Medical Center, Leiden, The Netherlands and subsequently did a post-doc at the University of California, Davis, after which she became an assistant professor, first at MD Anderson Cancer Center in Houston, TX, but she soon moved back to the Leiden University Medical center to join the department of Clinical Chemistry and Laboratory Medicine.
Authorship: L. Renee Ruhaak, F.P.H.T.M Romijn, N.P.M. Smit, M. Pieterse, A. Van der Laarse, C.M. Cobbaert
Leiden University Medical Center, department of Clinical Chemistry and Laboratory Medicine
| Short Abstract Apolipoprotein (a) (Apo(a)) is believed to be an independent risk factor for cardiovascular disease. Because of its size polymorphism, quantitation of apo(a) is inherently challenging, but the LC-MS technique has potential for accurate quantitation of apo(a) irrespective of its size polymorphisms. We here present the addition of medium abundant serum apo(a) to our previously developed test for absolute quantitation of high abundant serum apolipoproteins A1, B, C-I, C-II, C-III and E. By including an SPE step to enhance sensitivity, we were able to reach total CV’s below 5% for the quantitative peptide LFLEPTQADIALLK, while maintaining desirable precision for the high abundant apolipoproteins. Value-assigned matrix based serum pools were used for Apo(a) calibration, and a method comparison to an immunoassay based method showed results were exchangeable with mean bias of 4%. |
Long Abstract
Introduction
There is currently strong evidence for a causal relationship between concentrations of lipoprotein (a) (lp(a)) particles and cardiovascular disease, independent of the traditional lipid based CVD marker (HDL, LDL, TG and tC) [1]. Lp(a) consists of an LDL-like particle, which is covalently bound to one apolipoprotein (a) (apo(a)) molecule. Therefore, quantitation of apo(a) is believed to be important for CVD risk assessment. Apo(a) comprises a number of repetitive segments, the kringle IV-2 repeats, which gives rise to size heterogeneity, affecting all immunoassay based tests [2]. Here we present the extension of our previously developed multiplex apolipoprotein test [3] with medium abundant apo(a), focusing on accurate quantitation of apo(a) irrespective of its number of kringle IV-2 repeats.
Methods
A tryptic digest of lp(a) purified from human plasma was prepared to assess potential peptides for quantitation, and proteotypic peptides as well as kringle-specific peptides were selected based on empirical data as well as MRM atlas, the online tool PeptidePicker and literature. Eight peptides were synthesized and transitions for these peptides were developed and optimized using RP-HPLC-QQQ-MS. Because we aimed to include the measurement of apo(a) in the already existing multiplexed method for quantitation of apolipoproteins A1, B, C-I, C-II, C-III and E [3], the digestion efficiency was evaluated, and an SPE step was included to reach sufficient sensitivity. Matrix based calibrators were targeted and precision and bias of the developed test were evaluated using CLSI EP-15 protocol.
Results
Based on the lp(a) digest and the in-silico peptide selection tools, eight peptides from apo(a) were selected, and transitions could be established for all eight peptides. However, when these peptides were immediately assessed in serum, like we used to do for the other apolipoproteins [3], the sensitivity of the test was too low. An SPE step using Oasis HLB stationary phase was optimized to ensure accurate recoveries for all peptides, which increased the sensitivity of the test more than 20-fold. The digestion efficiencies were assessed and shown to plateau within 3 hours digestion time for 6 out of the 8 peptides, indicating their suitability for inclusion in our previously developed protocol. An EP-15 protocol was used to assess precision, in which CV’s below 5% were reached for the quantitative peptide LFLEPTQADIALLK as well as the kringle IV-2 specific peptide GTYSTTVTGR, while maintaining desirable precision for the high abundant apolipoproteins. CVs of 15% or higher were obtained for the other apo(a) peptides, indicating these peptides are less suitable for accurate quantitation. Value-assigned matrix based serum pools were used for Apo(a) calibration, and a method comparison to an immunoassay based method showed results were exchangeable with mean bias of 4%.
Conclusions & Discussion
We here present the extension of our previously developed multiplex qCCP test [3] for high abundant serum apolipoproteins A1, B, C-I, C-II, C-III and E with the inclusion of medium abundant polymorphic apo(a). The method allows for quantitation of apo(a) independent of apo(a)s size polymorphism, and will aid to better understand the role of apo(a) concentration in cardiovascular disease risk assessment.
References & Acknowledgements:
1. Nordestgaard BG, Langsted A. Lipoprotein (a) as a cause of cardiovascular disease: insights from epidemiology, genetics, and biology. J Lipid Res 2016;57:1953-1975.
2. Marcovina SM, Albers JJ. Lipoprotein (a) measurements for clinical application. J Lipid Res 2016;57:526-37.
3. van den Broek I, Romijn FP, Nouta J, van der Laarse A, Drijfhout JW, Smit NP, et al. 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;62:188-97.
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