Proteomic approaches for the identification and quantification of clinically relevant biomarkers

Abstract

Cardiovascular diseases are a significant health issue affecting people worldwide. Early diagnosis of cardiovascular diseases is crucial for the successful treatment of conditions such as heart attacks and for preventing death. Cardiac troponin I (cTnI) is a vital biomarker for the diagnosis and risk assessment of heart attacks. In healthy individuals, cTnI levels are found below 45 nanograms per liter. During and in the hours following a heart attack, it is released into the blood stream due to heart tissuedamage, leading to an increase in its levels. cTnI can be detected in the blood in various forms, including a ternary complex (cTnC-cTnT-cTnI), a binary complex (cTnI-cTnC), and free forms. It is highly susceptible to proteolysis and enzymatic changes. Consequently, various forms of cTnI, including proteolyzed, phosphorylated, oxidized, and reduced forms, can be found in the blood. All these variables lead to differences in cTnI measurements. There are many cTnI tests available on the market. The different variations circulating in the blood can be recognized by different monoclonal antibodies specific to different epitopes of cTnI. The various versions of cTnI, along with the different antibodies used, have increased the correlations between commercial tests more than tenfold, yet standardization remains challenging. Laboratories use different clinical decision thresholds depending on the test used. Different assay cutoffs have the potential to confuse physicians, leading to the misinterpretation of cTnI results; hence, there is urgency for cTnI standardization. The standardization and/or harmonization of cTnI assays is considered a high priority by the International Consortium for Harmonization of Clinical Laboratory results (ICHCLR). According to ISO 17511, the standardization of the measurement of a biomarker requires a metrological traceability chain. This chain begins with a primary reference measurement procedure (RMP), which assigns quantity values to a primary reference material (RM). Primary RMs are used to assign values to a secondary RM. With this secondary RM, values are assigned to working and product calibrators for routine quantification of the biomarker in patient samples. This traceability chain allows the values reported for patient care to be traced back to the International System of Units (SI). In this way, metrological traceability supports the long-term stability and comparability of routine laboratory measurement results. The standardization or harmonization of cTnI measurement requires the development of RMs and RMPs. The traceability chain proposed by the International Federation of Clinical Chemistry and Laboratory Medicine Working Group on Standardization of Troponin I (IFCC WG-TNI) incorporates all these standardization steps. One of the tasks that the IFCC working group is focused on to establish the proposed traceability chain is the development of a higher-order RMP. In this thesis, the focus has been on developing two different analytical methods to support the development of a RMP. Both analytical procedures involve targeted and bottom-up proteomic approaches. In both methods, isotope dilution mass spectrometry (IDMS) has been used to determine the absolute amount of cTnI. For quantifying proteins using the IDMS method, two different strategies have been employed: the protein-based calibration strategy and the peptide-based calibration strategy. Each method has its own advantages and disadvantages. The first developed analytical method allows for the determination of cTnI from human serum using a protein-based calibration strategy. In this context, human cardiac troponin complex material (NIST SRM 2921) has been selected for use as a calibrant. The troponin complex was purified from human heart tissue and consists of three subunits: troponin T (cTnT), troponin I (cTnI), and troponin C (cTnC). As an internal standard, isotopically labeled cTnI protein with the same sequence as cTnI has been used. To extract cTnI from a complex matrix like serum, an immunoaffinity enrichment strategy has been employed. As the first step of immunoaffinity enrichment, two different diameters of magnetic particles were selected: micro (Dynabeads® MyOne™, 1 μm) and nano (Nanomag®-D, 130 nm). A monoclonal antibody capable of binding to cTnI was immobilized on both types of magnetic nanoparticles, and their cTnI enrichment efficiencies were compared. Magnetic nanoparticles (Nanomag®-D, 130 nm) were chosen for further experiments because the peak areas of two selected tryptic peptides of cTnI were relatively higher. Next, the maximum loading capacity of the magnetic nanoparticles was determined. It was found that when 100 μg of antibody was added to 1 mg of particles, 59.2 ± 5.7 μg/mg of antibody could be bound. Using the synthesized nanoparticle-antibody conjugate, the required amount for cTnI enrichment from 1 ml of serum was calculated, and it was determined that 10 μl of conjugate was sufficient to capture all cTnI in 1 ml of serum for analysis. As a result of these optimizations, the isotope dilution liquid chromotograpy tandem mass spectrometry (ID-LC-MS/MS) method using the developed protein-based calibration strategy allows the measurement of cTnI in the range of 0.6 to 24 μg/L (R > 0.996). The limit of quantification (LOQ) was determined to be 1.8 μg/L, and the limit of detection (LOD) was 0.6 μg/L. Intermediate precision was found to be below 9.6%, and repeatability ranged from 2.0% to 8.7% for all quality control materials. The accuracy of the analyzed quality control materials was between 90% and 110%. Total measurement uncertainties (n=6) were found to be below 12.5% for all levels. The second developed ID-LC-MS/MS method allows the determination of cTnI in human serum using a peptide-based calibration strategy. In this method, two tryptic peptides (TLLLQIAK and NITEIADLTQK) of cTnI were selected and synthesized as calibrants. Isotopically labeled versions of the selected peptides were used as internal standards. Peptide impurity correction amino acid (PICAA) analysis was performed to assign values to the synthetic peptides, thereby producing SI-traceable primary peptide standards. Peptide-based calibration approach also employed two surrogate matrices to construct the calibration curve. The surrogate matrices were evaluated based on parameters such as linearity, accuracy, repeatability, intermediate precision, and trueness. It was observed that both matrices yielded similar results, indicating consistency in their performance. To ensure complete cleavage of the cTnI protein and enhance proteolysis yield, optimizations such as trypsin digestion methods, enzyme-to-protein ratio, and digestion time were performed. The best trypsin cleavage yield was obtained using the Filter-Aided Sample Preparation (FASP) method with a 1:10 enzyme-to-protein ratio and overnight digestion. The developed analytical method using the peptide-based calibration strategy enables the quantitative determination of cTnI in the range of 0.6–21.6 μg/L. Intermediate precision RSD was less than 28.9%, and repeatability RSD was less than 10% across all concentration levels. The recovery rate ranged between 72% and 151%. Four patient serum samples with suspected heart attack were measured using the developed method, and the results showed discrepancies of more than 50% compared to those obtained with immunoassay. Finally, the performance of the peptide-based calibration strategy was compared with the protein-based measurement strategy. In conclusion, this thesis has developed two different ID-LC-MS/MS methods using a targeted and bottom-up proteomic approaches. Both methods were compared with each other in terms of effectiveness. This efforts aim to support the metrology community in adopting new approaches and developing SI-traceable peptide and protein primary standards and/or reference procedures tailored to specific needs. Standardization and harmonization of cTnI across laboratories are undeniably complex tasks. However, the IFCC WG-TNI believes that cTnI measurement is standardizable. Given the critical role of cTnI in patient management, the significant effort invested is worthwhile. The proposed measurement methods will play a role in supporting the activities of the IFCC WG-TNI. These studies are necessary and logical steps towards the harmonization of results obtained from different test kits.