LEE- Moleküler Biyoloji-Genetik ve Biyoteknoloji-Doktora
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Yazar "Karagüler, Nevin Gül" ile LEE- Moleküler Biyoloji-Genetik ve Biyoteknoloji-Doktora'a göz atma
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ÖgeBacterial cellulose production using enzymatic hydrolysate of olive pomace(Graduate School, 2023-10-25) Öztan Sağdıç, Ceren ; Karagüler, Nevin Gül ; Tüter, Melek ; 521152107 ; Molecular Biology-Genetics and BiotechnologyBacterial cellulose is a biopolymer which has the identical chemical and structural properties with plant cellulose. However, it is recently of great scientific interest due to its superior characteristics such as high degree of polymerization, water retention capacity, and biocompatibility, compared to its plant-originated counterpart. Additionally, while plant cellulose is often bound to lignin, pectin and hemicellulose polymers, BC is obtained as a pure product. By virtue of these features, bacterial cellulose has been successfully employed in many applications among which are food, paper, biosensors, electronics, drug delivery systems, cosmetics, and wound healing materials. Along with some bacteria genera such as Acetobacter, Pseudomonas, Rhizobium and Escherichia, this polymer can also be formed by some algae (Valonia, Chaetamorpha) and fungi. It has been considered to procure the microorganisms an evolutionary advantage by protecting them against environmental factors such as UV radiation, desiccation and, contamination. There are several standardized growth media for the production of bacterial cellulose. Nonetheless, high cost of these laboratory-grade ingredients obstructs the large scale use of bacterial cellulose. Heretofore, numerous strategies have been proposed to minimize the medium cost. Molasses, rotten fruit, orange peel can be counted among the alternative media which gave promising results for bacterial cellulose generation. In this study, use of lignocellulosic material, particularly of olive pomace as carbon source for bacterial cellulose production was demonstrated. Lignocellulose, being the most abundant biopolymer in the world, has a great potential to serve as a substitute for fossil fuels. In fact, bioethanol and biofuel generation from lignocellulose by means of microorganims has been widely applied in industry. Similarly, in this work, it was aimed to obtain monomeric sugars from lignocellulose which in turn was used as carbon source for the cellulose producing bacteria- Novacetimonas hansenii. On the other hand, olive pomace (OP) is a by-product of the olive oil industry. Olive pomace is made up of olive pulp, stones, and skin of the fruit. It may pose environmental problems if not conducted properly. Approximately 35 - 45 kg dry olive pomace is obtained from 100 kg olive during oil production. This material is generally sorted and burned for energy production Nevertheless, it has been shown that OP is high in lignocellulosic content and the need for a more efficient way to use this material is apparent. In the first part of the study, two strains were compared according to their cellulose production yields. In addition, several cultivation conditions were performed to determine the most effective method. N. hansenii (ATCC 53582) in static growth condition gave the best results and therefore applied on the next steps. Moreover, three agricultural waste products; meat-bone flour, fish flour, and olive pomace were investigated for their efficiency to function as growth media. While no cellulose was formed with meat-bone flour and fish flour media, little cellulose was obtained in the medium prepared with olive pomace and lactose. After that, several trials with the use of olive pomace as nitrogen source while examining the performance of lactose and glucose as carbon sources were realized. However, elementary analysis revealed that the nitrogen content of olive pomace was not sufficient to supply the growth medium as nitrogen source. Besides, cellulose produced in olive pomace medium had poor mechanical qualities which is not suitable for any further application. On the other hand, olive pomace was shown to possess high organic component with approximately 45 % carbon. Therefore, in the second part of the study, generation of monomeric sugars by degradation of the lignocellulose of olive pomace was aimed. For this, acidic pretreatment and enzymatic hydrolysis were applied respectively. Acidic pretreatment breaks down the complex organization of lignocellulosic material to expose cellulose and hemicellulose for enzymatic degeneration. Subsequent enzymatic hydrolysis generates monomeric sugars which can be used by microorganisms. In this study, olive pomace was pretreated with 1 % phosphoric acid at 170 oC at 8 bar in order to separate cellulose from hemicellulose and lignin, thus unveiling the hydrolysable ends and producing oligosaccharides. Consecutive enzymatic reaction was conducted at 50 oC for 72 h with enzyme:substrate concentrations varying from 1.5 to 30 % (w/w) in static and agitated conditions. The reducing sugar concentration of the liquid part following the acidic pretreatment was determined by glucose hexokinase assay and found to be too low to lead to any microbial growth. Furthermore, reducing sugar concentration of each enzymatic hydrolysate was detected by dinitrosalicylic acid (DNSA) assay. Among varying enzyme:substrate concentrations, 30 % enzyme reaction in static condition resulted in the highest reducing sugar yield with 9.31 g/l. Enzymatic hydrolysates were scanned for the presence of galactose, glucose, mannose, arabinose, xylose, rhamnose, lactose, fructose, maltose and cellobiose by HPLC and distribution of these sugars along different hydrolysates was determined. For each hydrolysate, glucose was found to be the major monosaccharide. Growth media were prepared from selected hydrolysates with the ingredients of Hestrin-Schramm medium, except the carbon source, however, no cellulose formed. Therefore, the hydrolysates were detoxified to eliminate the inhibitory molecules generated in course of pretreatment. Among the methods attempted, Ca(OH)2 treatment was shown to be the most effective. From the media prepared with detoxified hydrolysates, the highest amount of bacterial cellulose production was 0.68 g/l. In addition, Hestrin-Schramm conventional medium and the medium with enzymatic hydrolysates were compared according to the substrate conversion ratio, cellulose production rate and yield. Sugar consumption in the control and test media was also detected. In the third part of the study, bacterial cellulose produced in the alternative medium was characterized with X-ray Diffraction Analysis, Fourier-Transform Infrared Spectroscopy (FTIR), and Scanning Electron Microscopy (SEM). X-ray diffraction demonstrated that the new BC posssesed the typical cellulose I peaks. However, the BC had a small amount of phosphate salts which caused HAP signals on the diffratogram. The FTIR analysis showed that the new bacterial cellulose had the characteristic spectrum of cellulose and no impurities were found. Similarly, with SEM analysis, it was demonstrated that the new material had nano-sized fibrillary structure very similar to the control material.
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ÖgeDiscovery of novel enzymes using proteomic approaches(Graduate School, 2021-01-26) Kılınç Öztuğ, Merve ; Karagüler, Nevin Gül ; Akgöz, Müslüm ; 521142107 ; Molecular Biology-Genetics and BiotechnologyThermophilic microorganisms that survive and grow in extreme environments, above temperatures of 50 °C, have been well studied over the last decade allowing us to increase our knowledge of the compositional and functional potential of these microbial communities. These microorganisms are of great importance for industrial processes since they express heat-resistive enzymes with the potential to serve as a biocatalyst in the future. Developing proteomic and metaproteomic approaches to discover novel enzymes from environmental samples is growing research of interest owing to the advanced mass spectrometry (MS) based techniques. In this study, proteomics and metaproteomics approaches were applied to discover novel enzymes from harsh environmental conditions. Geothermal sources are among the habitats of thermophilic bacteria. In Turkey, there are many spas that have the potential habitat for numerous thermophilic bacteria, and this offers a good opportunity for the discovery of new thermophilic microorganisms. In this study, a thermophilic bacterial consortium of the Armutlu Hot Spring in the Yalova region of Turkey was investigated in a culture-dependent manner using proteomic approaches.
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ÖgeExploration of novel serine protease do-like HtrA from acigöl(Graduate School, 2023-12-06) Kılıç, Meryem Menekşe ; Karagüler, Nevin Gül ; Balcı Çelik, Nurgül ; 521112113 ; Molecular Biology-Genetics and BiotechnologyEnzymes involved in industrial biotechnological processes take place in conditions of extremely high temperature, high pH, and high salinity or when there are organic solvents that have made it necessary to discover enzymes resistant to these conditions. Microorganisms in extreme environments adapt to varying levels of stress such as very high pH, temperature, salt, and pressure. For the last 20 years, researchers have focused especially on extreme environments for the discovery of enzymes that are resistant to extreme conditions, with the hypothesis that the enzymes of microorganisms adapted to these conditions can also work under extreme conditions. In this context, microorganisms can be isolated from their environment and their enzymes can be characterized by traditional microbiological methods. Besides this, new enzymes have been discovered by the method called 'metagenomics', which is not based on culture. Environments with high salt concentration are divided into two in terms of their ionic compositions. Many high salt concentration environments were formed by the evaporation of seawater, also called 'thalassohaline'. Their salt content is similar to seawater and the pH varies from basic to slightly acidic. Environments with high salt concentrations, called 'Athalassohaline', are completely different from seawater in terms of ionic composition. Acıgöl, which is our study area, is a lake with high salt concentration, which is included in the 'Athalassohaline' state group. In this study, samples from Acıgöl were employed. Acıgöl is located between the provincial borders of Denizli and Burdur in the Aegean Region of our country. Looking at the chemical composition of the lake, it is seen that Na+, K+, Cl−, and SO4 2− ions are dominant. The salinity of the Acıgöl changes between 5.8%-13%, pH between 7.8-8.2, and temperature varies seasonally between 8 °C and 32 °C. These changing extreme conditions force the microorganisms in the lake to cellular and enzymatic adaptation. These organisms adapted to high salt concentration are called 'Halophilic' microorganisms, meaning salt-loving. Based on this information, the main subject of the study is the discovery of enzymes of halophilic microorganisms that can be used in difficult industrial processes. The primary objective of this study is to obtain new proteases, which are of industrial importance, by function-based screening of culturable microorganisms. In line with this goal, firstly, soil samples taken from Acıgöl were diluted in Nutrient Broth and spread on nutrient agar petri dishes containing 10% NaCl and 1% skim milk, and the species containing protease activity were determined. It was determined by the transparent region around the colonies that the isolate had protease activity, resulting from the breakdown of skim milk. With this screening method, halophilic species in Acıgöl, which actively produce protease, were determined. Sixmorphologically different species were determined. Twoshowed protease activity, and the species with t huge zones were chosen for further studies. In the second part of the study, the whole genome of the determined species was sequenced with the New Generation Sequencing method (Illumina HiSeq 2500 platform), and its serine proteases and other biotechnologically potential enzymes were determined. According to the sequencing results, it was determined that the entire genome of the isolated species was 4,708.499 bp (base pair) in length, had a G+C ratio of 36.66%, and had 4536 gene-coding sequences. In addition, it was revealed that 99.81% ratio similarity to Virgibacillus marismortui species according to 16S rDNA sequence similarity. The whole-genome average nucleotide identity (ANI) value was obtained as 99.44% and digital DNA-DNA hybridization was computed as 88.8%. The average amino acid identity ratio (AAI: Average Amino acid Identity) was calculated as 98.69%. In addition to genomic analyses, the isolated species was also examined phenotypically and biochemically. It was determined that the species was gram positive (Gr+), both alkaliphilic and moderately halophilic, motile, endospore-forming, and protease-producing bacterium. The isolated strain shows optimum growth at 37 °C with salinity and pH ranging from 5-10% and 6 and 9, respectively. As a result of this polyphasic analysis, it was conclueded that the isolate was a subspecies of Virgibacillus species, and it has been brought to the literature with the name Virgibacillus sp. AGTR. All genome information is stored in the NCBI database. Accession number JAJERH000000000. The last step of the study aimed to produce by recombinantly and characterize the serine protease from a new isolate. Among the four serine proteases determined by whole genome analysis, the Serine protease Do-like HtrA with the lowest sequence similarity rate and fewer studies in the literature was selected for recombinant production. The Serine protease Do-like HtrA is a member of the Trypsin-like serine protease superfamily (Tryp_SPc Superfamily) and S1-C subfamily. HtrA (high-temperature requirement A), a periplasmic heat-shock protein, it has two different functions. While it shows molecular chaperone properties at low temperatures, it shows proteolytic activity at high temperatures. The structure of this kind of protease differs slightly from other commercial and well-studied proteases. Due to these properties, it could be used specifically in the pharmaceutical industry. For the recombinant production of Serine protease Do-like HtrA, primers that contain EcoR I and SacI restriction sites were designed to be specific to the start and end sequences of the gene of interest (targeting the 5' and 3' ends). By using the genome of the isolated Virgibacillus sp. AGTR strain as a template, the target protease gene was amplified and ligated into the pET-28-a(+)expression vector. The cloned vector was inserted into E. coli BL21, E. coli C43 (DE3), and RosettaTM 2 expression cells to determine the best expression host cell. As a result of the purification study, the RosettaTM 2 cell was selected for expression. Expression studies were performed with 0.1 mM, 0.5 mM, and, 1 mM IPTG concentrations at 30 ºC and 37 ºC for up to 6 hours. The highest level of expression was achieved with 0.1 mM IPTG in 4 hours at 30 °C. Successfully expressed protease gene was purified by the His-tag method. The estimated molecular weight of the protein was 42100 Da and the isoelectric point was 4.53 which is calculated using the ExPASy program. As a result of purification, the molecular weight of the enzyme (42.1 kDa) was compatible with the predicted value, according to SDS-PAGE and Western blot tests.
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ÖgeProtein engineering applications on industrially important enzymes(Fen Bilimleri Enstitüsü, 2019) Özgün, Gülşah ; Karagüler, Nevin Gül ; Moleküler Biyoloji-Genetik ve Biyoteknoloji ; Molecular Biology-Genetics and BiotechnologyBüyüyen biyoteknoloji marketi, beraberinde biyokatalizörlerin geliştirilmesi ve yeni özellikler kazandırılmasına yönelik çalışmaların artmasına yol açmıştır. Endüstriyel ortamlar genellikle doğal enzimlerin kullanımı için uygun olmayan ekstrem şartlar barındırmaktadır, yüksek sıcaklık, basınç ve çok yüksek veya düşük pH gibi koşullarda, çoğunlukla doğal enzimler istenilen performansı sağlayamamaktadır. Başta protein mühendisliği olmak üzere birçok disiplin farklı endüstriyel alanlarda enzimlerin etkili bir şekilde kullanımına yönelik stratejiler geliştirmektedirler. Protein mühendisliği, endüstriyel sektörün özelliklerine ve enzimin kullanılacağı ortama bağlı olarak istenilen özelliklerin geliştirilmesinde, örneğin aktivite ve stabilitenin arttırılması, substrat veya koenzimin spesifisitesinin değiştirilmesi veya geliştirilmesi, optimum pH nın değiştirilmesi gibi stratejilere yönelik rasyonel tasarım, yönlendirilmiş evrim ve kombinasyonel uygulamaları kullanmaktadır. Protein mühendisliği stratejilerinin belirlenmesinde aynı zamanda enzime ait (yapı-fonksiyon) bilgilerin varlığı veya yokluğu etkili olmaktadır. Protein mühendisliği, moleküler biyoloji temeline dayanarak proteinin yapı fonksiyon ilişkisinin anlaşılmasına olanak sağlayarak, proteinin genetik düzeyde yeniden dizayn edilmesini ve istenilen özellikte biyokatalizörlerin oluşturulmasını mümkün kılar. Bu kapsamda tez, endüstriyel öneme sahip Bacillus subtilis lipazA (bsLipA) ve Candida methylica format dehidrogenaz (cmFDH) enzimlerinin üç farklı stratejinin kullanıldığı protein mühendisliği uygulamalarına odaklanmıştır. i) Optimum pH nın ii) ve koenzim spesifitesinin değiştirilmesi iii) termal stabilitenin arttırılması hedeflenen stratejiler olmuştur. Deneysel çalışmalar üç bölümde detaylandırılmıştır. Tezin birinci kısmında, bsLipA enziminin optimum pH'sının değiştirilmesi amacıyla rasyonel tasarım uygulamalarından bölgeye özel mutasyon yöntemi uygulanmıştır. Lipazlar (EC 3.1.1.3) trigliseridlerin serbest yağ asitleri ve gliserole hidrolizini gerçekleştirirlerken, aynı zamanda transesterifikasyon, aminoliziz ve asidoliziz reaksiyonlarını da katalizlerler. Mikrobiyal lipazların susuz ve az-sulu ortamlardaki potansiyeli, enzimin çok yönlü biyoteknolojik bir araç haline gelmesini sağlamıştır. Farklı endüstriyel alanlarda uygulama imkanı bulan lipazlar, son yıllarda sıvı veya süperkritik karbon dioksit (LCO2/SCCO2) gibi hijyen sistemlerinde hidrolitik enzim katkısı olarak kullanım potansiyeline sahiptir. LCO2/SCCO2 sistemlerinin temizleme etkisinin hidrolitik enzim ilavesi ile arttırılması mümkün olmasına karşın, çözgen olarak kullanılacak olan LCO2 / SCCO2' in polar olmayan bir çözgen olması ve düşük su içeriği sebebiyle, kullanılacak enzim sisteminin az sulu ve düşük pH' ya sahip çözgen sistemlerinde aktif olması beklenmektedir. Bacillus subtilis lipaz (bsLipA), geniş bir pH (4-11) aralığına sahip olmasına karşın optimum pH'sı 10'dur, bu sebeple LCO2 / SCCO2 çözgen sistemlerinde kullanımı için modifikasyonu gerekmektedir. Bacillus subtilis lipaz A (PDB ID: 1ISP) kristal yapısı ve Insight II programı kullanılarak enzimin optimum pH değişimini sağlayacak hedef mutantlar belirlenmiştir. Proteinin aktif bölgesinde katalitik özellikteki amino asitlerin pKa değerlerini etkileyebileceği düşünülen, yaklaşık 9 Å'luk mesafe içerisinde olan ve katalitik amino asitler ile doğrudan ilişkili olan amino asitlerin, bölgeye özel mutasyon tekniği ile tekli G11E, N18R, L102R, G103R, G104R, I157R mutantları oluşturulmuştur. N18R ve G103R mutantlarının template olarak kullanılması ile ikili (G11E-N18R, G103R- N18R, G103R- G11E, and G103R- G104R) mutantların da, bölgeye özel mutasyon tekniği ile oluşturulması planlanmıştır. Tezin ikinci kısmı cmFDH enziminin koenzim spesifitesinin değiştirilmesine yönelik yarı-rasyonel tasarım uygulamalarından bölge saturasyon mutagenez yöntemini kapsamaktadır. Tezin üçüncü kısmında ise cmFDH enziminin termal stabilitesinin arttırılmasına yönelik, , rasyonel tasarım uygulaması olan bölgeye özel mutasyon yöntemi uygulanmıştır. NAD+-bağımlı format dehidrogenaz enzimi (EC 1.2.1.2, FDH), format iyonunun karbondiokside dönüşümünü katalizlerken, NAD+ molekülünün NADH'e indirgenmesini sağlamaktadır. Gerçekleşen reaksiyonun basitliği, kullanılabilirliği, düşük maliyeti, termodinamik özellikleri ve son ürün olan CO2' in reaksiyondan kolaylıkla uzaklaştırılabilmesi gibi avantajlarından dolayı FDH, kimya endüstrisindeki kiral bileşiklerin sentezi için çok önemli olan NAD(P)H rejenerasyonunda potansiyel bir sistemdir. Fakat, doğada bulunan FDH'lerin çoğunluğunun NAD+ koenzimine spesifik olması ve düşük termal stabiliteye sahip olması, FDH'in kullanımını kısıtlamaktadır. Bu sınırlandırmaların aşılması amacıyla, tezin ikinci kısmında, cmFDH enziminin koenzim spesifitesinin değiştirilmesi için koenzim bağlama bölgesinde, koenzim spesifitesinden sorumlu amino asitler, Pseodomonas. sp.101 ve Candida boidinii FDH kristal yapıları baz alınarak, Insight II (Accelrys) programı ile oluşturulan cmFDH homoloji modeli yardımıyla belirlenmiştir. Belirlenen D195, Y196 ve Q197 bölgelerine ait dejenere primerler ile uygulanan bölge saturasyon mutagenez çalışması sonucunda her bölge için mutant kütüphaneleri oluşturulmuştur. NADP+ koenzimi ile aktivite gösteren aday mutantlar kolorimetrik tarama metoduyla belirlenmiştir. İki nesil oluşturulan adayların protein üretimi ve saflaştırılması neticesinde NADP+ koenzimine karşı olan ilgisi test edilmiştir. Yapılan çalışma sonucunda ikili mutantlardan D195S / Q197T ve D195S / Y196L mutant cmFDH enzimlerinin NADP+` ye karşı katalitik etkinlikleri yabanıl tip cmFDH enzimine kıyasla, sırasıyla 56000 ve 50000 kat artmıştır. Çalışmanın son kısmında, cmFDH enziminin termal stabilitesinin geliştirilmesi amacıyla, protein yüzeyindeki esnekliği yüksek olan oyuklar ve bu oyuklarda bulunan esnek amino asit kalıntıları hedef alınmıştır. Pseodomonas. sp.101 ve Candida boidinii FDH kristal yapıları baz alınarak, ExPASy programı ile cmFDH homoloji modeli elde edilmiştir. Oluşturulan model üzerinde FIRST algoritması kullanılarak, proteinin esnek oyukları ve hedef amino asit kalıntıları belirlenmiştir. Yapılan bilgisayar çalışmaları neticesinde belirlenen 12 aday; M131A, V133I, V139W, P140R, D158N, I162V, F186L, V219M, F247A, E272W, R277N ve K301R bölgeye özel mutasyon yöntemi ile oluşturulmuştur. Mutant adayların protein üretimi ve saflaştırılması neticesinde gerçekleştirilen kinetik ve sıcaklık çalışmaları sonucunda, birinci oyukta yer alan M131A mutant enziminin, rekombinant yabani tip cmFDH enzimine kıyasla yarı ömründe 4 °C'lik bir artışla diğer mutantlar arasında en iyi termal stabilite profili gösterdiği belirlenmiştir. Bu tez kapsamında yapmış olduğumuz tüm çalışmalar, endüstriyel kullanım için enzimlerin modifikasyonunda, doğru stratejiler kullanıldığında protein mühendisliği uygulamalarının, başarı sağladığını göstermektedir.
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ÖgeProteomic approaches for the identification and quantification of clinically relevant biomarkers(Graduate School, 2024-10-16) Küçük Aşıcıoğlu, Meltem ; Karagüler, Nevin Gül ; Kılınç Öztuğ, Merve ; 521192111 ; Molecular Biology-Genetics and BiotechnologyCardiovascular 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.