Douwe de Boer

Posttranslational modifications of cardiac troponin T: An overview

Cardiac troponin (cTn) is an important sarcomeric protein complex situated on the thin filament and is involved in the regulation of cardiac muscle contraction. This regulation is primarily controlled by Ca(2+) binding to troponin C and in addition fine-tuned by the posttranslational modification of cTnI and cTnT. The vast majority of cTnT modifications involve the phosphorylation by protein kinase C (PKC) or other kinases and the N-terminal cleavage by caspase and calpain. In vitro studies employing reconstituted detergent-skinned fiber bundles and cell culture generally show a detrimental effect of cTnT phosphorylation on muscle contraction, which is backed by some in vivo studies finding increased cTnT phosphorylation in heart failure, but contradicted by others. In addition, N-terminal cleavage of cTnT is thought to be another factor influencing cardiac contraction. Time-dependent degradation of cTnT has been observed in human serum upon myocardial infarction. These molecular changes might influence the immunoreactivity of cTnT in the clinical immunoassay and have consequences for the clinical interpretations of these measurements. No consensus has yet been reached on the occurrence and extent of these observations and their underlying processes are subject of intense scientific debate. This review will focus on discussing these modifications, their implications on physiology and disease and summarizes the complex interplays of different enzymes on the molecular forms of cTnT and their associated effects.

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

UNLABELLED 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.

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.

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.

Large Variation in Measured Cardiac Troponin T Concentrations after Standard Addition in Serum or Plasma of Different Individuals

To the Editor: During acute myocardial infarction (AMI), 1 cardiac troponin T (cTnT) is released from the damaged myocardium. One would expect a strong correlation between concentrations of cTnT measured in the blood after AMI and the extent of myocardial damage. However, various studies have shown a rather moderate correlation between infarct size and cTnT concentrations (1, 2). Frequently considered explanations for this phenomenon are methodological or focused on physiological factors associated with differences in release and elimination of cTnT (2). In contrast, less attention has been paid to the possible presence of inherent factors in the blood, such as (auto)antibodies or proteases, that might modify cTnT or interfere with the assay. We hypothesized that these factors may be ubiquitously present in the general population and might affect the measured concentrations of cTnT in individuals. We measured cTnT, cTnI, creatine kinase MB isoenzyme (CK-MB), and myoglobin concentrations in sera from 24 healthy volunteers (age range 22–54 years) before and after the addition of serum from a patient suffering from AMI (age 59 years, cTnT concentration approximately 19.5 μg/L). All participants gave written informed consent and leftover material was used in accordance with the code of proper secondary use of human tissue …

Thrombin Activation via Serum Preparation is Not the Root Cause for Cardiac Troponin T Degradation

To the Editor: We read with interest the recent article by Katrukha et al. concerning thrombin-mediated degradation of human cardiac troponin T (cTnT) in serum (1). In their study, Katrukha and coworkers confirmed the finding of Streng et al. that human coagulation factor II (thrombin) is a strong mediator of cTnT degradation (2). They compared cTnT degradation in serum and heparin plasma collected simultaneously from the same patients with acute myocardial infarction (AMI), revealing substantial differences in cTnT fragment composition. They also performed in vitro experiments in which troponin T, thrombin, and hirudin were supplemented in various combinations. In addition, the cleavage site in cTnT was localized by use of mass spectrometry and probing of different proteolytic fragments with the use of various antibodies. Their main conclusion was that the primary 29-kDa cTnT fragment was mainly formed because of thrombin activation during serum preparation. We believe that the evidence for thrombin as one of the main proteases for cTnT degradation is convincing. However, we disagree with their final conclusion because there is published evidence suggesting …

Gelofusine® affects the quality control performance of QuickVue® point-of-care human chorionic gonadotropin test devices

OBJECTIVE To characterize the influence of Gelofusine (succinylated gelatin) on the performance of point-of-care testing (POCT) for human chorionic gonadotropin (hCG) devices. DESIGN AND METHODS Three brands of urine and urine/serum hCG POCT devices were verified. RESULTS Succinylated gelatin affected the performance of the control band in the QuickVue hCG POCT devices. Alternative devices were not affected. The hCG test performance is not influenced. CONCLUSIONS Gelofusine affects specifically the quality control performance of the QuickVue hCG POCT devices.

Implications of matrix adducts to protein analyte ions for surface‐enhanced laser desorption/ionization and matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometric analysis in clinical chemistry

Besides the introduction of liquid chromatography/tandem mass spectrometry (LC/MS/MS), also that of matrixassisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) has opened an analytical window, which expands the possibilities in clinical chemistry to establish diagnoses. The main advantage of MALDITOFMS is obtaining molecular mass information in a simple way through the m/z value of MHþ ions. Combined with sophisticated sample preparation, MALDI-TOFMS has even become one of the technologies of interest in new-venture research for biomarkers in clinical chemistry. The studies of transthyretin (TTR) variants are in that respect typical examples of establishing diagnoses in cancer research using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOFMS). Other routine-like clinical applications are the MALDI-TOFMS analyses of haemoglobin (Hb) variants studying inherited haemoglobinpathies and posttranslational modifications, or of albumin in albuminuria. Like every analytical technology, MALDI-TOFMS has its limitations and interpretation problems. Certain problems are caused by so-called matrix adduct ions, as illustrated in the application of TTR analysis. These adduct ions originate from thematrix and their presencemay result in an analytical interfering signal. Matrices are required to perform MALDITOFMS and play a key role in the formation of MHþ ions, although the exact mechanisms are still disputed. The formation of matrix adduct ions has already been recognized directly after the development of MALDI-TOFMS. It has been described that certain matrix molecules can lose a neutral moiety such as water during the MALDI process and that, subsequently, a dehydrated matrix molecule (ma– H2O) may attach to a MH þ ion of a protein analyte and form the adduct ion [MHþ(ma–H2O)]. When applying sinapinic acid (SA) for the MALDI-TOFMS analysis of apomyoglobin, that adduct ion is in that respect a wellknown example. In this study we describe the phenomenonofmatrix adduct ions in specific cases ofMALDI-TOFMS analysis of the microheterogeneity of TTR variants and the profiling ofHb variants in haemoglobinopathies in particular. Also the MALDI-TOFMS analysis of albumin will be discussed. The relevance of the phenomenon ofmatrix adduct ions is thatdependingon themass-resolving capabilities of the instrument used: adduct ions might limit the possibilities of establishing a diagnosis in clinical chemistry by MALDITOFMS. Especially, intact [MHþma]þ anddehydratedmatrix adduct [MHþ(ma–H2O)] ions may cause problems, which obviously should be taken into account. MALDI-TOFMS analysis in our study was performed either with a PBS IIc analyzer (Ciphergen Biosystems, CA, USA), which is actually an axial extraction linear TOF instrument, or with a QSTARXLQ-TOFmass spectrometer (Applied Biosystems/MDS Sciex, Concord, ON, Canada), which is an orthogonal extraction reflectron TOF instrument. The PBS IIc analyzer is a typical instrument as applied in SELDI-TOFMS analysis with a maximum scan range up to m/z 500000, while the QSTAR XL analyzer is a multipurpose instrument, which in the MALDI-single TOFMS mode has a maximum scan range up tom/z 40000. Both mass spectrometers are equippedwith a 337 nm nitrogen laser. We collected for our analyses a total number of 60–120 spectra per spot measurement. After smoothing we calibrated externally the PBS IIc spectra according to the manufacturer’s recommendations. The QSTAR XL spectra were calibrated internally according to the manufacturer’s recommendation using the MHþ ion of cytochrome C and apomyoglobin at m/z 12231.92 and 16952.51, respectively. Reference standards of bovine heart cytochrome C and equine skeletal muscle myoglobin were obtained from Sigma (St. Louis, MO, USA). The matrices used were SA and acyano-4-hydroxycinnamic acid (CHCA) both from Fluka (Buchs, Switzerland). Spots were prepared by applying 1mL of solution of interest (analyte and internal calibrants) to the surface of a gold chip array (Ciphergen Biosystems) or to the surface of a QSTAR XL stainless steel sample plate (Applied Biosystems/MDS Sciex), where the solution was allowed to dry. For the gold chip array and unless specified the residues were covered with 0.6mL of SA mixture: a solution of 56mM of SA and 50mM of ammonium dihydrogen phosphate Ultrex in a mixture of 100% acetonitrile and 1% trifluoroacetic anhydride (TFAA) in Milli-Q water (1:1; v/v). After drying, the step with 0.6mL of SA mixture was repeated. If specified a CHCA mixture was employed: a solution of 106mM CHCA and 50mM ammonium dihydrogen phosphate Ultrex in a mixture of 100% acetonitrile and 1% TFAA inMilli-Qwater (1:1; v/v). For the stainless steel sample plate the residues were covered with 0.6mL of SA mixture with a different solvent composition: a solution of 56mM of SA and 50mM of ammonium dihydrogen phosphate Ultrex in a mixture of 100% acetonitrile and 0.2% TFAA inMilli-Q water (1:2; v/v). After drying, the step with 0.6mL of SA mixture was also repeated. The solvent composition was different because of the spreading out properties on stainless steel. In all cases spots were always analyzedwithin some hours after drying. The use of TFAA (Sigma) instead of trifluoroacetic acid was preferred because the anhydride contains potentially less alkali metal ion contaminations. For a similar reason ammonium dihydrogen phosphate (J.T. Baker, Deventer, The Netherlands) was used, which is known to suppress alkali metal ion adduct clusters. I: 10.1002/rcm.4304