Differences in Cardiac Troponin T Composition in Myocardial Infarction and End-Stage Renal Disease Patients: A Blood Tube Effect?

BACKGROUND: Cardiac troponin T (cTnT) is key in diagnosing myocardial infarction (MI) but is also elevated in end-stage renal disease (ESRD) patients. Specific larger cTnT proteoforms were identified for the acute phase of MI, while in serum of ESRD patients solely small cTnT fragments were found. However, others allocated this to a pre-analytic effect due to abundant thrombin generation in serum. Therefore, we investigated the effect of various anticoagulation methods on cTnT composition and concentration and compared the cTnT composition of MI and ESRD patients. METHODS: The agreement of cTnT concentrations between simultaneously collected serum, lithium-heparin (LH) plasma, and ethylenediaminetetraacetic acid (EDTA) plasma was studied using the high-sensitivity (hs-)cTnT immunoassay. cTnT proteoform composition was investigated in a standardized time-dependent manner through spike experiments and in simultaneously collected blood matrixes of MI and ESRD patients. RESULTS: Excellent hs-cTnT concentration agreements were observed across all blood matrixes (slopes > 0.98; 95% CI, 0.96-1.04). Time-dependent degradation (40 kDa intact:29 kDa fragment:15 to 18 kDa fragments) was found in LH plasma and EDTA plasma, and serum in ratios (%) of 90:10:0, 0:5:95, and 0:0:100, respectively (48 h after blood collection). Moreover, gel filtration chromatography (GFC) profiles illustrated mainly larger cTnT proteoforms in MI patients, while in ESRD patients mainly 15 to 18 kDa fragments were found for all matrices. CONCLUSIONS: The extent of cTnT degradation in vitro is dependent on the (anti)coagulation method, without impacting hs-cTnT concentrations. Furthermore, mainly larger cTnT proteoforms were present in MI patients, while in ESRD patients mainly small 15 to 18 kDa cTnT fragments were found. These insights are essential when developing a novel hs-cTnT assay targeting larger cTnT proteoforms.

A Low or High Physical Activity Level Does Not Modulate Prostate Tumor Tissue Protein Synthesis Rates.

INTRODUCTION: Physical activity level has been identified as an important factor in the development and progression of various types of cancer. In this study, we determined the impact of a low versus high physical activity level on skeletal muscle, healthy prostate, and prostate tumor protein synthesis rates in vivo in prostate cancer patients. METHODS: Thirty prostate cancer patients (age, 66 ± 5 yr; body mass index, 27.4 ± 2.9 kg·m -2 ) were randomized to a low (<4000 steps per day, n = 15) or high (>14,000 steps per day, n = 15) physical activity level for 7 d before their scheduled radical prostatectomy. Daily deuterium oxide administration was combined with the collection of plasma, skeletal muscle, nontumorous prostate, and prostate tumor tissue during the surgical procedure to determine tissue protein synthesis rates throughout the intervention period. RESULTS: Daily step counts averaged 3610 ± 878 and 17,589 ± 4680 steps in patients subjected to the low and high physical activity levels, respectively ( P < 0.001). No differences were observed between tissue protein synthesis rates of skeletal muscle, healthy prostate, or prostate tumor between the low (1.47% ± 0.21%, 2.74% ± 0.70%, and 4.76% ± 1.23% per day, respectively) and high (1.42% ± 0.16%, 2.64% ± 0.58%, and 4.72% ± 0.80% per day, respectively) physical activity group (all P > 0.4). Tissue protein synthesis rates were nearly twofold higher in prostate tumor compared with nontumorous prostate tissue. CONCLUSIONS: A short-term high or low physical activity level does not modulate prostate or prostate tumor protein synthesis rates in vivo in prostate cancer patients. More studies on the impact of physical activity level on tumor protein synthesis rates and tumor progression are warranted to understand the potential impact of lifestyle interventions in the prevention and treatment of cancer.

Low Levels of Serum and Intracellular Vitamin C in Hospitalized COVID-19 Patients.

Vitamin C is a crucial micronutrient for human immune cell function and has potent antioxidant properties. It is hypothesized that vitamin C serum levels decline during infection. However, the precise mechanisms remain unknown. To gain deeper insights into the true role of vitamin C during infections, we aimed to evaluate the body's vitamin C storage during a SARS-CoV-2 infection. In this single-center study, we examined serum and intracellular vitamin C levels in peripheral blood mononuclear cells (PBMCs) of 70 hospitalized COVID-19 patients on the first and fifth days of hospitalization. Also, clinical COVID-19 severity was evaluated at these timepoints. Our findings revealed a high prevalence of hypovitaminosis C and vitamin C deficiency in hospitalized COVID-19 patients (36% and 15%). Moreover, patients with severe or critical disease exhibited a higher prevalence of low serum vitamin C levels than those with moderate illness. Serum vitamin C levels had a weak negative correlation with clinical COVID-19 severity classification on the day of hospitalization; however, there was no correlation with intracellular vitamin C. Intracellular vitamin C levels were decreased in this cohort as compared to a healthy cohort and showed further decline during hospitalization, while serum levels showed no relevant change. Based on this observation, it can be suggested that the reduction of intracellular vitamin C may be attributed to its antioxidative function, the need for replenishing serum levels, or enhanced turnover by immune cells. These data give an incentive to further investigate the role of intracellular vitamin C in a larger and more heterogeneous cohort as well as the underlying mechanisms.

No Effect of Vitamin C Administration on Neutrophil Recovery in Autologous Stem Cell Transplantation for Myeloma or Lymphoma: A Blinded, Randomized Placebo-Controlled Trial.

Vitamin C is an important micronutrient for various immune cells. It increases phagocytic cell function and is necessary for T and natural killer (NK) cell development. Patients in need of an autologous hematopoietic stem cell transplantation (HSCT) are often vitamin C-depleted. We therefore hypothesized that vitamin C supplementation could improve immune recovery in autologous HSCT patients. This blinded, placebo-controlled trial included 44 patients randomized to receive vitamin C or a placebo. The following outcome measures used were clinical and immunological parameters, among others: time to neutrophil recovery, serum, and intracellular vitamin C values. Twenty-one patients received vitamin C, and 23 received a placebo. The time to neutrophil recovery did not differ between the two groups at 11.2 days (p = 0.96). There were no differences in hospitalization time (19.7 vs. 19.1 days, p = 0.80), the incidence of neutropenic fever (57% vs. 78%, p = 0.20), or 3-month overall survival (90.5% vs. 100%, p = 0.13). Bacteremia seemed to occur less in the vitamin C group (10% vs. 35%, p = 0.07). Our study shows no benefit from vitamin C supplementation on neutrophil recovery and hospitalization, despite possible lower rates of bacteremia in the vitamin C group. Therefore, we do not advise vitamin C supplementation in this treatment group.

Cardiac Troponin T: The Impact of Posttranslational Modifications on Analytical Immunoreactivity in Blood up to the Excretion in Urine.

Cardiac troponin T (cTnT) is a sensitive and specific biomarker for detecting cardiac muscle injury. Its concentration in blood can be significantly elevated outside the normal reference range under several pathophysiological conditions. The classical analytical method in routine clinical analysis to detect cTnT in serum or plasma is a single commercial immunoassay, which is designed to quantify the intact cTnT molecule. The targeted epitopes are located in the central region of the cTnT molecule. However, in blood cTnT exists in different biomolecular complexes and proteoforms: bound (to cardiac troponin subunits or to immunoglobulins) or unbound (as intact protein or as proteolytic proteoforms). While proteolysis is a principal posttranslational modification (PTM), other confirmed PTMs of the proteoforms include N-terminal initiator methionine removal, N-acetylation, O-phosphorylation, O-(N-acetyl)-glucosaminylation, N(ɛ)-(carboxymethyl)lysine modification and citrullination. The immunoassay probably detects several of those cTnT biomolecular complexes and proteoforms, as long as they have the centrally targeted epitopes in common. While analytical cTnT immunoreactivity has been studied predominantly in blood, it can also be detected in urine, although it is unclear in which proteoform cTnT immunoreactivity is present in urine. This review presents an overview of the current knowledge on the pathophysiological lifecycle of cTnT. It provides insight into the impact of PTMs, not only on the analytical immunoreactivity, but also on the excretion of cTnT in urine as one of the waste routes in that lifecycle. Accordingly, and after isolating the proteoforms from urine of patients suffering from proteinuria and acute myocardial infarction, the structures of some possible cTnT proteoforms are reconstructed using mass spectrometry and presented.

Multi-Site Coronary Vein Sampling Study on Cardiac Troponin T Degradation in Non-ST-Segment-Elevation Myocardial Infarction: Toward a More Specific Cardiac Troponin T Assay.

Background Cardiac troponin T ( cTnT ) is seen in many other conditions besides myocardial infarction, and recent studies demonstrated distinct forms of cTnT . At present, the in vivo formation of these different cTnT forms is incompletely understood. We therefore performed a study on the composition of cTnT during the course of myocardial infarction, including coronary venous system sampling, close to its site of release. Methods and Results Baseline samples were obtained from multiple coronary venous system locations, and a peripheral artery and vein in 71 non- ST -segment-elevation myocardial infarction patients. Additionally, peripheral blood was drawn at 6- and 12-hours postcatheterization. cTnT concentrations were measured using the high-sensitivity- cTnT immunoassay. The cTnT composition was determined via gel filtration chromatography and Western blotting in an early and late presenting patient. High-sensitivity - cTnT concentrations were 28% higher in the coronary venous system than peripherally (n=71, P<0.001). Coronary venous system samples demonstrated cT n T-I-C complex, free intact cTnT , and 29 kD a and 15 to 18 kD a cTnT fragments, all in higher concentrations than in simultaneously obtained peripheral samples. While cT n T-I-C complex proportionally decreased, and disappeared over time, 15 to 18 kD a cTnT fragments increased. Moreover, cT n T-I-C complex was more prominent in the early than in the late presenting patient. Conclusions This explorative study in non- ST -segment-elevation myocardial infarction shows that cTnT is released from cardiomyocytes as a combination of cT n T-I-C complex, free intact cTnT , and multiple cTnT fragments indicating intracellular cTnT degradation. Over time, the cT n T-I-C complex disappeared because of in vivo degradation. These insights might serve as a stepping stone toward a high-sensitivity- cTnT immunoassay more specific for myocardial infarction.

Leucine coingestion augments the muscle protein synthetic response to the ingestion of 15 g of protein following resistance exercise in older men.

Older adults have shown an attenuated postexercise increase in muscle protein synthesis rates following ingestion of smaller amounts of protein compared with younger adults. Consequently, it has been suggested that older adults require the ingestion of more protein to increase postexercise muscle protein synthesis rates compared with younger adults. We investigated whether coingestion of 1.5 g of free leucine with a single 15-g bolus of protein further augments the postprandial muscle protein synthetic response during recovery from resistance-type exercise in older men. Twenty-four healthy older men (67 ± 1 yr) were randomly assigned to ingest 15 g of milk protein concentrate (MPC80) with (15G+LEU; n = 12) or without (15G; n = 12) 1.5 g of free leucine after performing a single bout of resistance-type exercise. Postprandial protein digestion and amino acid absorption kinetics, whole body protein metabolism, and postprandial myofibrillar protein synthesis rates were assessed using primed, continuous infusions with l-[ring-2H5]phenylalanine, l-[ring-2H2]tyrosine, and l-[1-13C]leucine combined with ingestion of intrinsically l-[1-13C]phenylalanine-labeled milk protein. A total of 70 ± 1% (10.5 ±0.2 g) and 75 ± 2% (11.2 ± 0.3 g) of the protein-derived amino acids were released in the circulation during the 6-h postexercise recovery phase in 15G+LEU and 15G, respectively (P < 0.05). Postexercise myofibrillar protein synthesis rates were 16% (0.058 ± 0.003 vs. 0.049 ± 0.002%/h, P < 0.05; based on l-[ring-2H5]phenylalanine) and 19% (0.071 ± 0.003 vs. 0.060 ± 0.003%/h, P < 0.05; based on l-[1-13C]leucine) greater in 15G+LEU compared with 15G. Leucine coingestion further augments the postexercise muscle protein synthetic response to the ingestion of a single 15-g bolus of protein in older men.

Dose-Dependent Increases in Whole-Body Net Protein Balance and Dietary Protein-Derived Amino Acid Incorporation into Myofibrillar Protein During Recovery from Resistance Exercise in Older Men.

BACKGROUND: Age-related decline in skeletal muscle mass is at least partly attributed to anabolic resistance to food intake. Resistance exercise sensitizes skeletal muscle tissue to the anabolic properties of amino acids. OBJECTIVE: The present study assessed protein digestion and amino acid absorption kinetics, whole-body protein balance, and the myofibrillar protein synthetic response to ingestion of different amounts of protein during recovery from resistance exercise in older men. METHODS: Forty-eight healthy older men [mean ± SEM age: 66 ± 1 y; body mass index (kg/m2): 25.4 ± 0.3] were randomly assigned to ingest 0, 15, 30, or 45 g milk protein concentrate after a single bout of resistance exercise consisting of 4 sets of 10 repetitions of leg press and leg extension and 2 sets of 10 repetitions of lateral pulldown and chest press performed at 75-80% 1-repetition maximum. Postprandial protein digestion and amino acid absorption kinetics, whole-body protein metabolism, and myofibrillar protein synthesis rates were assessed using primed, continuous infusions of l-[ring-2H5]-phenylalanine, l-[ring-2H2]-tyrosine, and l-[1-13C]-leucine combined with ingestion of intrinsically l-[1-13C]-phenylalanine and l-[1-13C]-leucine labeled protein. RESULTS: Whole-body net protein balance showed a dose-dependent increase after ingestion of 0, 15, 30, or 45 g of protein (0.015 ± 0.002, 0.108 ± 0.004, 0.162 ± 0.008, and 0.215 ± 0.009 µmol Phe · kg-1 · min-1, respectively; P < 0.001). Myofibrillar protein synthesis rates were higher after ingesting 30 (0.0951% ± 0.0062%/h, P = 0.07) or 45 g of protein (0.0970% ± 0.0062%/h, P < 0.05) than after 0 g (0.0746% ± 0.0051%/h). Incorporation of dietary protein-derived amino acids (l-[1-13C]-phenylalanine) into de novo myofibrillar protein showed a dose-dependent increase after ingestion of 15, 30, or 45 g protein (0.0171 ± 0.0017, 0.0296 ± 0.0030, and 0.0397 ± 0.0026 mole percentage excess, respectively; P < 0.05). CONCLUSIONS: Dietary protein ingested during recovery from resistance exercise is rapidly digested and absorbed. Whole-body net protein balance and dietary protein-derived amino acid incorporation into myofibrillar protein show dose-dependent increases. Ingestion of ≥30 g protein increases postexercise myofibrillar protein synthesis rates in older men. This trial was registered at Nederlands Trial Register as NTR4492.

Brain tissue plasticity: protein synthesis rates of the human brain.

All tissues undergo continuous reconditioning via the complex orchestration of changes in tissue protein synthesis and breakdown rates. Skeletal muscle tissue has been well studied in this regard, and has been shown to turnover at a rate of 1-2% per day in vivo in humans. Few data are available on protein synthesis rates of other tissues. Because of obvious limitations with regard to brain tissue sampling no study has ever measured brain protein synthesis rates in vivo in humans. Here, we applied stable isotope methodology to directly assess protein synthesis rates in neocortex and hippocampus tissue of six patients undergoing temporal lobectomy for drug-resistant temporal lobe epilepsy (Clinical trial registration: NTR5147). Protein synthesis rates of neocortex and hippocampus tissue averaged 0.17 ± 0.01 and 0.13 ± 0.01%/h, respectively. Brain tissue protein synthesis rates were 3-4-fold higher than skeletal muscle tissue protein synthesis rates (0.05 ± 0.01%/h; P < 0.001). In conclusion, the protein turnover rate of the human brain is much higher than previously assumed.

Mass Spectrometric Identification of Cardiac Troponin T in Urine of Patients Suffering from Acute Myocardial Infarction.

BACKGROUND: Because of its high cardiospecificity, cardiac troponin T (cTnT) is one of the first-choice biomarkers to diagnose acute myocardial infarction (AMI). cTnT is extensively fragmented in serum of patients suffering from AMI. However, it is currently unknown whether all cTnT is completely degraded in the body or whether some cTnT fragments can leave the body via urine. The aim of the present study is to develop a method for the detection of cTnT in urine and to examine whether cTnT is detectable in patient urine. METHODS: Proteins in urine samples of 20 patients were precipitated using a cTnT-specific immunoprecipitation technique and a nonspecific acetonitrile protein precipitation. After in-solution digestion of the precipitated proteins, the resulting peptides were separated and analyzed using HPLC and mass spectrometry with a targeted selected ion monitoring assay with data-dependent tandem mass spectrometry (t-SIM/dd-MS2). RESULTS: The t-SIM/dd-MS2 assay was validated using a synthetic peptide standard containing 10 specific cTnT peptides of interest and with purified human intact cTnT spiked in urine from healthy individuals. Using this assay, 6 different cTnT-specific peptides were identified in urine samples from 3 different patients, all suffering from AMI. CONCLUSIONS: We show here for the first time that cTnT can be present in the urine of AMI patients using a targeted LC-MS/MS assay. Whether the presence of cTnT in urine reflects a physiological or pathophysiological process still needs to be elucidated.

Food ingestion in an upright sitting position increases postprandial amino acid availability when compared with food ingestion in a lying down position.

Dietary protein digestion and absorption kinetics determine the postprandial increase in muscle protein synthesis. We recently demonstrated that body position during feeding can modulate the postprandial rise in plasma amino acid availability. Here we investigated whether protein ingestion in an upright sitting body position accelerates gastric emptying and improves dietary protein digestion and subsequent amino acid absorption compared with feeding in a supine lying body position. In a crossover design, 8 young males (age, 26 ± 1 years; body mass index, 24.0 ± 0.9 kg·m-2) ingested 20 g intrinsically l-[1-13C]-phenylalanine-labeled milk protein plus 1.5 g paracetamol while sitting in an upright position or lying down in a supine position. Blood samples were collected frequently during a 5-h postprandial period. Gastric emptying rates and dietary protein digestion and absorption were assessed using plasma paracetamol and amino acid concentrations as well as plasma l-[1-13C]-phenylalanine enrichments. Peak plasma leucine concentrations were higher when protein was ingested in an upright sitting versus lying position (213 ± 15 vs 193 ± 12 µmol·L-1, P < 0.05), which was accompanied by a trend for a greater overall leucine response (13 989 ± 720 vs 11 875 ± 1073 AU, respectively; P = 0.05). Peak plasma paracetamol concentrations were higher in the sitting versus lying treatment (11.6 ± 0.5 vs 9.3 ± 0.6 mg·L-1, P < 0.05). Protein ingestion in an upright sitting position accelerates gastric emptying and increases the postprandial rise in plasma amino acid availability by increasing protein digestion and amino acid absorption rates. Therefore, feeding in an upright body position as opposed to a lying position is an important prerequisite to allow proper postprandial muscle protein accretion.

Identification and Characterization of Cardiac Troponin T Fragments in Serum of Patients Suffering from Acute Myocardial Infarction.

BACKGROUND: Cardiac troponin T (cTnT) is the preferred biomarker for the diagnosis of acute myocardial infarction (AMI). It has been suggested that cTnT is present predominantly in fragmented forms in human serum following AMI. In this study, we have used a targeted mass spectrometry assay and epitope mapping using Western blotting to confirm this hypothesis. METHODS: cTnT was captured from the serum of 12 patients diagnosed with AMI using an immunoprecipitation technique employing the M11.7 catcher antibody and fractionated with SDS-PAGE. Coomassie-stained bands of 4 patients at 37, 29, and 16 kDa were excised from the gel, digested with trypsin, and analyzed on a Q Exactive instrument set on targeted Selected Ion Monitoring mode with data-dependent tandem mass spectrometry (MS/MS) for identification. Western blotting employing 3 different antibodies was used for epitope mapping. RESULTS: Ten cTnT peptides of interest were targeted. By using MS/MS, all of these peptides were identified in the 37-kDa, intact, cTnT band. In the 29- and 16-kDa fragment bands, 8 and 4 cTnT-specific peptides were identified, respectively. Some of these peptides were "semitryptic," meaning that their C-termini were not formed by trypsin cleavage. The C-termini of these semitryptic peptides represent the C-terminal end of the cTnT molecules present in these bands. These results were confirmed independently by epitope mapping. CONCLUSIONS: Using LC-MS, we have succeeded in positively identifying the 29- and 16-kDa fragment bands as cTnT-derived products. The amino acid sequences of the 29- and 16-kDa fragments are Ser79-Trp297 and Ser79-Gln199, respectively.

Cardiac troponin T degradation in serum is catalysed by human thrombin.

Cardiac troponin T (cTnT) has been shown to be present in fragmented forms in human serum after acute myocardial infarction (AMI). While calpain-1 and caspase-3 have been identified as intracellular proteases able to cleave the N-terminus of cTnT, it is still unclear which proteases are responsible for the extensive and progressive cTnT fragmentation observed in serum of AMI-patients. In this pilot study we have investigated the possibility that human thrombin may be involved in this process. Purified human cTnT was spiked in unprocessed and deproteinated serum in the presence or absence of either purified human thrombin or PPACK thrombin inhibitor. After immunoprecipitation, SDS-PAGE and Western blotting we observed an increase in cTnT fragmentation when purified thrombin was added to deproteinated serum. Consequently, the addition of thrombin inhibitor to unprocessed serum resulted in a decrease of cTnT fragmentation. Our results suggest that multiple enzymes are involved in cTnT degradation, and that thrombin plays an important role.

Increasing Insulin Availability Does Not Augment Postprandial Muscle Protein Synthesis Rates in Healthy Young and Older Men.

CONTEXT: Skeletal muscle protein synthesis is highly responsive to food intake. It has been suggested that the postprandial increase in circulating insulin modulates the muscle protein synthetic response to feeding. OBJECTIVE: The objective of the study was to investigate whether a greater postprandial rise in circulating insulin level increases amino acid uptake in muscle and augments postprandial muscle protein synthesis rates. PARTICIPANTS AND DESIGN: Forty-eight healthy young (age 22 ± 1 y; body mass index 22.0 ± 0.3 kg/m2) and older males (age 68 ± 1 y; body mass index 26.3 ± 0.4 kg/m2) ingested 20 g intrinsically L-[1-13C]-leucine- and L-[1-13C]-phenylalanine-labeled casein protein with or without local insulin infusion. Primed continuous infusions of L-[1-13C]-leucine and L-[ring-2H5]-phenylalanine were applied, with arterial and venous blood samples and muscle biopsies being collected during a 5-hour postprandial period. RESULTS: Insulin administration did not increase overall leg blood flow (P = .509) but increased amino acid uptake over the leg in both young and older subjects (P = .003). The greater amino acid uptake over the leg did not further increase postprandial muscle protein synthesis rates (0.050% ± 0.006% and 0.037% ± 0.004% per hour vs 0.044% ± 0.004% and 0.037% ± 0.002% per hour in the insulin-stimulated vs control condition in the young and older groups, respectively; P = .804) and did not affect postprandial deposition of dietary protein-derived amino acids in de novo muscle protein (P = .872). CONCLUSION: Greater postprandial plasma insulin availability stimulates amino acid uptake over the leg but does not further augment postprandial muscle protein synthesis rates or stimulate the postprandial deposition of protein derived amino acids into de novo muscle protein in healthy young and older men.

Ascorbic acid serum levels are reduced in patients with hematological malignancies.

In this paper we demonstrate that patients treated with chemotherapy and/or hematopoietic stem cell transplantation (HSCT) have highly significant reduced serum ascorbic acid (AA) levels compared to healthy controls. We recently observed in in vitro experiments that growth of both T and NK cells from hematopoietic stem cells is positively influenced by AA. It might be of clinical relevance to study the function and recovery of immune cells after intensive treatment, its correlation to AA serum levels and the possible effect of AA supplementation.

Validation, optimisation, and application data in support of the development of a targeted selected ion monitoring assay for degraded cardiac troponin T.

Cardiac troponin T (cTnT) fragmentation in human serum was investigated using a newly developed targeted selected ion monitoring assay, as described in the accompanying article: "Development of a targeted selected ion monitoring assay for the elucidation of protease induced structural changes in cardiac troponin T" [1]. This article presents data describing aspects of the validation and optimisation of this assay. The data consists of several figures, an excel file containing the results of a sequence identity search, and a description of the raw mass spectrometry (MS) data files, deposited in the ProteomeXchange repository with id PRIDE: PXD003187.

Development of a targeted selected ion monitoring assay for the elucidation of protease induced structural changes in cardiac troponin T.

Cardiac troponin T (cTnT) is a highly cardiospecific protein commonly used in the diagnosis of acute myocardial infarction (AMI), but is subject to proteolytic degradation upon its release in the circulation. In this study, a targeted mass spectrometry assay was developed to detect peptides which are differentially present within the different degradation products. cTnT was spiked in human serum and incubated at 37 °C to induce proteolytic degradation. Isolation and fractionation of cTnT and its fragments from serum were performed using immunoprecipitation and SDS-PAGE. Bands migrating to 37 kDa (intact cTnT), 29 kDa (primary fragment), and 19, 18, and 16kDa (secondary fragments) were excised, digested, and subsequently analysed using targeted selected ion monitoring on a UHPLC-coupled quadrupole-Orbitrap mass spectrometer. Sixteen precursor ions from a total of 11 peptides unique to cTnT were targeted. Precursor ions were detectable up until 1200 ng/L cTnT, which is a typical cTnT concentration after AMI. With tandem-MS and relative quantification, we proved the formation of cTnT fragments upon incubation in human serum and identified differentially present peptides in the fragment bands, indicative of N- and C-terminal proteolytic cleavage. These findings are of importance for the development of future cTnT assays, calibrators, and quality control samples. BIOLOGICAL SIGNIFICANCE: In this study we have developed a gel-based targeted mass spectrometry assay which is able to differentiate between different molecular forms of cTnT. The unravelling of the molecular presentation of cTnT in human serum is of importance in the field of clinical chemistry, where this highly specific and sensitive biomarker is being measured on a routinely basis in patient samples. Knowledge of the amino acid sequence of the different cTnT fragments may aid in the development of improved calibrators and quality control samples. In addition, different fragmentation patterns may be indicative of different underlying pathologies. New antibodies for future assays targeting specific areas of cTnT can thus be created based on this information. This assay will be used in future experiments to assess the fragmentation pattern of cTnT in serum of multiple patient groups in our laboratory.