Lipoprotein(a), also known as Lp(a), is an LDL-like particle composed of apolipoprotein(a) (apo(a)) bound covalently to apolipoprotein B100. Plasma concentrations of Lp(a) are highly heritable and vary widely between individuals. Elevated plasma concentration of Lp(a) is considered as an independent, causal risk factor of cardiovascular disease (CVD). Targeted mass spectrometry (LC-SRM/MS) combined with stable isotope-labeled recombinant proteins provides robust and precise quantification of proteins in the blood, making LC-SRM/MS assays appealing for monitoring plasma proteins for clinical implications. This study presents a novel quantitative approach, based on prototypic peptides, to determine the absolute concentration of apo(a) from two microliters of plasma and qualified according to guideline requirements for targeted proteomics assays. After optimization, assay parameters such as linearity, lower limits of quantification (LLOQ), intra-assay variability (CV: 4.72%) and inter-assay repeatability (CV: 7.76%) were determined and the LC-SRM/MS results were benchmarked against a commercially available immunoassay. In summary, the measurements of an apo(a) single copy specific peptide and a K4 specific peptide allows for the determination of molar concentration and relative size of apo(a) in individuals.

A pool of SIS PrESTs was made and spiked to empty wells of a 96 well LoBind plate (0030129512, Eppendorf, Hamburg, Germany). The plate was vacuum centrifuged to dry off any liquid. Ten times diluted plasma corresponding to two microliters of raw plasma was added to the plate of dried standards and the samples were digested following the same procedure as described. Each digest was then subjected to SPE using StageTips. Approximately 10 µg of peptide amount was loaded onto an Ultimate 3000 (Thermo Fischer Scientific, Waltham, MA, USA) LC-system fitted with a 15 cm EasySpray analytical column (PN ES802A rev.2, particle size: 2 µm, pore size: 100Å, 150 µm x 15 cm, Thermo Fischer Scientific) and an Acclaim PepMap 100 trap column (PN 160454, particle size: 5 µm, pore size: 100 Å, 0.3 mm x 5 mm, Thermo Fischer Scientific). The peptides were eluted across a linear gradient using a 35 min instrument with a flowrate of 3 µl/min with a mobile phase consisting of solvent A (3% ACN, 0.1% FA) and solvent B (95% ACN , 0.1% FA) and a gradient as described in Supplementary Table 4. The LC was coupled to a TSQ Altis (Thermo Fischer Scientific) operating with a cycle time of 0.5 seconds.

Plasma (K2-EDTA) for method development was collected from five healthy non-obese Caucasian volunteers, three female and two male donors, obtained from the AstraZeneca R&D Gothenburg biobank. Moreover, an additional ten randomly chosen human plasma samples also obtained from the AstraZeneca R&D Gothenburg biobank of healthy volunteers were used for the immunoassay measurements. Informed consent was obtained from all subjects. The study was performed according to local ethical regulations following approval from the regional ethics committee “Regionala Etikprövningsnämnden i Göteborg” in Gothenburg, Sweden. The plasma samples were pooled and aliquoted into 0.5 ml Protein LoBind Microcentrifuge Eppendorf tubes that were subsequently stored at -80°C and also used as quality control samples. A well-characterized healthy sample cohort (The Swedish Science for Life Laboratory SCAPIS Wellness Profiling (S3WP) was analyzed (38). Informed consent was obtained for all participants. The study was performed in accordance with the declaration of Helsinki and the study protocol was approved by the Ethical Review Board of Göteborg, Sweden (Regionala etikprövnignsnämnden, Gothenburg, Dnr 407-15, 2015-06-25). All samples were de-identified and randomized prior to the proteomics analysis.