RESUMO
Precise determination of circulating parathyroid hormone (PTH) concentration is crucial to diagnose and manage various disease conditions, including the chronic kidney disease-mineral and bone disorder. However, the lack of standardization in PTH assays is challenging for clinicians, potentially leading to medical errors because the different assays do not provide equivalent results and use different reference ranges. Here, we aimed to evaluate the impact of recalibrating PTH immunoassays by means of a recently developed LC-MS/MS method as the reference. Utilizing a large panel of pooled plasma samples with PTH concentrations determined by the LC-MS/MS method calibrated with the World Health Organization (WHO) 95/646 International Standard, five PTH immunoassays were recalibrated. The robustness of this standardization was evaluated over time using different sets of samples. The recalibration successfully reduced inter-assay variability with harmonization of PTH measurements across different assays. By recalibrating the assays based on the WHO 95/646 International Standard, we demonstrated the feasibility for standardizing PTH measurement results and adopting common reference ranges for PTH assays, facilitating a more consistent interpretation of PTH values. The recalibration process aligns PTH results obtained from various immunoassays with the LC-MS/MS method, providing more consistent and reliable measurements. Thus, establishing true standardization across all PTH assays is crucial to ensure consistent interpretation and clinical decision-making.
Assuntos
Distúrbio Mineral e Ósseo na Doença Renal Crônica , Insuficiência Renal Crônica , Humanos , Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem , Hormônio Paratireóideo , Insuficiência Renal Crônica/diagnósticoRESUMO
Cardiovascular diseases have cast a significant negative impact on the lives of millions worldwide. Over the years, extensive efforts have been dedicated to enhancing diagnostic and prognostic tools for these diseases. A growing body of evidence indicates that the angiotensin convertase enzyme (ACE) and the angiotensin convertase enzyme 2 (ACE2), and angiotensin peptide levels could hold a pivotal role in assisting clinicians with the management of cardiovascular conditions, notably hypertension and heart failure. However, despite the considerable body of knowledge in this domain, a void remains in the field of analytical methodologies for these molecules. In this study, we present a fully validated LC-MS/MS method for the precise quantitation of plasma angiotensin (1-7), (1-8), (1-9), and (1-10), following the guidelines set by the Clinical and Laboratory Standards Institute (CLSI). Our method not only enables the accurate quantification of angiotensin peptides but also provides a means to assess ACE and ACE2 activity. Remarkably, our method achieved a Lower Limit of Measurement Interval (LLMI) as low as 5 pg/mL. This has enabled the detection of angiotensin (1-7), (1-8), (1-9) and (1-10) and the accurate quantitation of angiotensin (1-7), (1-8) and (1-10) in all analyzed groups, including healthy controls, patients with high blood pressure, and patients with chronic kidney disease. To our knowledge, our method represents the most sensitive approach allowing for simultaneous quantitation of these four angiotensin peptides. A distinct advantage of our method, when compared to immunoassays, is its high sensitivity combined with comprehensive chromatographic separation of all currently known angiotensin peptides. This combination translates to an exceptional level of selectivity, underscoring the value and potential of our methodology in advancing cardiovascular disease research.
Assuntos
Doenças Cardiovasculares , Espectrometria de Massa com Cromatografia Líquida , Fragmentos de Peptídeos , Humanos , Cromatografia Líquida/métodos , Enzima de Conversão de Angiotensina 2 , Espectrometria de Massas em Tandem/métodos , Angiotensina I , Peptídeos , Angiotensina IIRESUMO
Since the late 1990s, cathepsin K cleavage sites in type I collagen have been extensively studied due to its ability to release bone resorption biomarkers such as CTX and NTX. However, gel-based methods and N-sequencing used in these studies lack sensitivity, especially for small to medium peptides. In this work, we propose a degradomics mass spectrometry-based workflow that combines protein digestion, Nano-LC-UDMSE, and several software tools to identify cathepsin K cleavage sites. This workflow not only identified previously known cleavage sites, but also discovered new ones. Multiple cleavage hotspots were found and described in type I α1 and type I α2 collagen, many of which coincided with pyridinoline crosslinks, known to stabilize the triple helix. Our results allowed us to establish a chronology of digestion and conclude that cathepsin K preferentially cleaves the extremities of type I collagen before the helical part. We also found that cathepsin K preferentially cleaves amino acid residues with long and hydrophobic lateral chains at the beginning of digestion, whereas no preferred amino acid residues were identified later in the digestion. In conclusion, our workflow successfully identified new cleavage sites and can be easily applied to other proteins or proteases.
Assuntos
Aminoácidos , Colágeno Tipo I , Catepsina K , Fluxo de Trabalho , Espectrometria de MassasRESUMO
Immunocapture is now a well-established method for sample preparation prior to quantitation of peptides and proteins in complex matrices. This short review will give an overview of some clinical applications of immunocapture methods, as well as protocols with and without enzymatic digestion in a clinical context. The advantages and limitations of both approaches are discussed in detail. Challenges related to the choice of mass spectrometer are also discussed. Top-down, middle-down, and bottom-up approaches are discussed. Even though immunocapture has its limitations, its main advantage is that it provides an additional dimension of separation and/or isolation when working with peptides and proteins. Overall, this short review demonstrates the potential of such techniques in the field of proteomics-based clinical medicine and paves the way for better personalized medicine.
Assuntos
Peptídeos , Proteínas , Humanos , Cromatografia Líquida/métodos , Espectrometria de Massas , Proteínas/análiseRESUMO
With the recent improvements in ion mobility resolution, it is now possible to separate small protomeric tautomers, called protomers. In larger molecules above 1000 Da such as peptides, a few studies suggest that protomers do exist as well and may contribute to their gas-phase conformational heterogeneity. In this work, we observed a CCS distribution that can be explained by the presence of protomers of surfactin, a small lipopeptide with no basic site. Following preliminary density functional theoretical calculations, several protonation sites in the gas phase were energetically favorable in positive ionization mode. Experimentally, at least three near-resolved IM peaks were observed in positive ionization mode, while only one was detected in negative ionization mode. These results were in good agreement with the DFT predictions. CID breakdown curve analysis after IM separation showed different inflection points (CE50) suggesting that different intramolecular interactions were implied in the stabilization of the structures of surfactin. The fragment ratio observed after collision-induced fragmentation was also different, suggesting different ring-opening localizations. All these observations support the presence of protomers on the cyclic peptide moieties of the surfactin. These data strongly suggest that protomeric tautomerism can still be observed on molecules above 1000 Da if the IM resolving power is sufficient. It also supports that the proton localization involves a change in the 3D structure that can affect the experimental CCS and the fragmentation channels of such peptides.
Assuntos
Peptídeos Cíclicos , Prótons , Lipopeptídeos , Conformação Molecular , Peptídeos Cíclicos/química , Subunidades Proteicas/químicaRESUMO
In the past, we developed a method inferring physicochemical properties from ion mobility mass spectrometry (IM-MS) data from polydisperse synthetic homopolymers. We extend here the method to biomolecules that are generally monodisperse. Similarities in the IM-MS behavior were illustrated on proteins and peptides. This allows one to identify ionic species for which intramolecular interactions lead to specific structures.
RESUMO
Disulfide bonds between cysteine residues are commonly involved in the stability of numerous peptides and proteins and are crucial for providing biological activities. In such peptides, the appropriate cysteine connectivity ensures the proper conformation allowing an efficient binding to their molecular targets. Disulfide bond connectivity characterization is still challenging and is a critical issue in the analysis of structured peptides/proteins targeting pharmaceutical or pharmacological utilizations. This study describes the development of new and fast gas-phase and in-solution electrophoretic methods coupled to mass spectrometry to characterize the cysteine connectivity of disulfide bonds. For this purpose, disulfide isomers of three peptides bearing two intramolecular disulfide bonds but different cysteine connectivity have been investigated. Capillary zone electrophoresis and ion mobility both coupled to mass spectrometry were used to perform the separation in both aqueous and gas phases, respectively. The separation efficiency of each technique has been critically evaluated and compared. Finally, theoretical calculations were performed to support and explain the experimental data based on the predicted physicochemical properties of the different peptides.
Assuntos
Cisteína/análise , Dissulfetos/química , Peptídeos/química , Eletroforese Capilar , Espectrometria de Mobilidade Iônica , Espectrometria de Massas , SoftwareRESUMO
When polymer mixtures become increasingly complex, the conventional analysis techniques become insufficient for complete characterization. Mass spectrometric techniques can satisfy this increasing demand for detailed sample characterization. Even though isobaric polymers are indistinguishable using simple mass spectrometry (MS) analyses, more advanced techniques such as tandem MS (MS/MS) or ion mobility (IM) can be used. Here, we report proof of concept for characterizing isomeric polymers, namely poly(2-n-propyl-2-oxazoline) (Pn-PrOx) and poly(2-isopropyl-2-oxazoline) (Pi-PrOx), using MS/MS and IM-MS. Pi-PrOx ions lose in intensity at higher accelerating voltages than Pn-PrOx ions during collision-induced dissociation (CID) MS/MS experiments. A Pn/i-PrOx mixture could also be titrated using survival yield calculations of either precursor ions or cation ejection species. IM-MS yielded shape differences in the degree of polymerization (DP) regions showing the structural rearrangements. Combined MS techniques are thus able to identify and deconvolute the molar mass distributions of the two isomers in a mixture. Finally, the MS/MS and IM-MS behaviors are compared for interpretation. Graphical Abstract .
RESUMO
Polymer characterizations are often performed using mass spectrometry (MS). Aside from MS and different tandem MS (MS/MS) techniques, ion mobility-mass spectrometry (IM-MS) has been recently added to the inventory of characterization technique. However, only few studies have focused on the reproducibility and robustness of polymer IM-MS analyses. Here, we perform collisional and electron-mediated activation of polymer ions before measuring IM drift times, collision cross-sections (CCS), or reduced ion mobilities (K0). The resulting IM behavior of different activated product ions is then compared to non-activated native intact polymer ions. First, we analyzed collision induced unfolding (CIU) of precursor ions to test the robustness of polymer ion shapes. Then, we focused on fragmentation product ions to test for shape retentions from the precursor ions: cation ejection species (CES) and product ions with m/z and charge state values identical to native intact polymer ions. The CES species are formed using both collision induced dissociation (CID) and electron transfer dissociation (ETD, formally ETnoD) experiments. Only small drift time, CCS, or K0 deviations between the activated/formed ions are observed compared to the native intact polymer ions. The polymer ion shapes seem to depend solely on their mass and charge state. The experiments were performed on three synthetic homopolymers: poly(ethoxy phosphate) (PEtP), poly(2-n-propyl-2-oxazoline) (Pn-PrOx), and poly(ethylene oxide) (PEO). These results confirm the robustness of polymer ion CCSs for IM calibration, especially singly charged polymer ions. The results are also discussed in the context of polymer analyses, CCS predictions, and probing ion-drift gas interaction potentials. Graphical Abstract.
RESUMO
Disulfide connectivity in peptides bearing at least two intramolecular disulfide bonds is highly important for the structure and the biological activity of the peptides. In that context, analytical strategies allowing a characterization of the cysteine pairing are of prime interest for chemists, biochemists, and biologists. For that purpose, this study evaluates the potential of MALDI in-source decay (ISD) for characterizing cysteine pairs through the systematic analysis of identical peptides bearing two disulfide bonds, but not the same cysteine connectivity. Three different matrices have been tested in positive and/or in negative mode (1,5-DAN, 2-AB and 2-AA). As MALDI-ISD is known to partially reduce disulfide bonds, the data analysis of this study rests firstly on the deconvolution of the isotope pattern of the parent ions. Moreover, data analysis is also based on the formed fragment ions and their signal intensities. Results from MS/MS-experiments (MALDI-ISD-MS/MS) constitute the last reference for data interpretation. Owing to the combined use of different ISD-promoting matrices, cysteine connectivity identification could be performed on the considered peptides. Graphical Abstract á .
Assuntos
Dissulfetos/análise , Peptídeos/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Sequência de Aminoácidos , Conotoxinas/química , Cisteína/análise , Isomerismo , Oxirredução , Espectrometria de Massas em Tandem/métodosRESUMO
Over the years, polymer analyses using ion mobility-mass spectrometry (IM-MS) measurements have been performed on different ion mobility spectrometry (IMS) setups. In order to be able to compare literature data taken on different IM(-MS) instruments, ion heating and ion temperature evaluations have already been explored. Nevertheless, extrapolations to other analytes are difficult and thus straightforward same-sample instrument comparisons seem to be the only reliable way to make sure that the different IM(-MS) setups do not greatly change the gas-phase behavior. We used a large range of degrees of polymerization (DP) of poly(ethylene oxide) PEO homopolymers to measure IMS drift times on three different IM-MS setups: a homemade drift tube (DT), a trapped (TIMS), and a traveling wave (T-Wave) IMS setup. The drift time evolutions were followed for increasing polymer DPs (masses) and charge states, and they are found to be comparable and reproducible on the three instruments. á .
RESUMO
Ion mobility (IM) is now a well-established and fast analytical technique. The IM hardware is constantly being improved, especially in terms of the resolving power. The Drift Tube (DTIMS), the Traveling Wave (TWIMS), and the Trapped Ion Mobility Spectrometry (TIMS) coupled to mass spectrometry are used to determine the Collision Cross-Sections (CCS) of ions. In analytical chemistry, the CCS is approached as a descriptor for ion identification and it is also used in physical chemistry for 3D structure elucidation with computational chemistry support. The CCS is a physical descriptor extracted from the reduced mobility (K0) measurements obtainable only from the DTIMS. TWIMS and TIMS routinely require a calibration procedure to convert measured physical quantities (drift time for TWIMS and elution voltage for TIMS) into CCS values. This calibration is a critical step to allow interinstrument comparisons. The previous calibrating substances lead to large prediction bands and introduced rather large uncertainties during the CCS determination. In this paper, we introduce a new IM calibrant (CCS and K0) using singly charged sodium adducts of poly(ethylene oxide) monomethyl ether (CH3O-PEO-H) for positive ionization in both helium and nitrogen as drift gas. These singly charged calibrating ions make it possible to determine the CCS/K0 of ions having higher charge states. The fitted calibration plots exhibit larger coverage with less data scattering and significantly improved prediction bands and uncertainties. The reasons for the improved CCS/K0 accuracy, advantages, and limitations of the calibration procedures are also discussed. A generalized IM calibration strategy is suggested.
RESUMO
Disulfide bonds are post-translationnal modifications that can be crucial for the stability and the biological activities of natural peptides. Considering the importance of these disulfide bond-containing peptides, the development of new techniques in order to characterize these modifications is of great interest. For this purpose, collision cross cections (CCS) of a large data set of 118 peptides (displaying various sequences) bearing zero, one, two, or three disulfide bond(s) have been measured in this study at different charge states using ion mobility-mass spectrometry. From an experimental point of view, CCS differences (ΔCCS) between peptides bearing various numbers of disulfide bonds and peptides having no disulfide bonds have been calculated. The ΔCCS calculations have also been applied to peptides bearing two disulfide bonds but different cysteine connectivities (Cys1-Cys2/Cys3-Cys4; Cys1-Cys3/Cys2-Cys4; Cys1-Cys4/Cys2-Cys3). The effect of the replacement of a proton by a potassium adduct on a peptidic structure has also been investigated. Graphical Abstract á .
Assuntos
Dissulfetos/análise , Espectrometria de Massas/métodos , Peptídeos/química , Sequência de Aminoácidos , Cisteína , PrótonsRESUMO
Disulfide bonds are post-translational modifications (PTMs) often found in peptides and proteins. They increase their stability toward enzymatic degradations and provide the structure and (consequently) the activity of such folded proteins. The characterization of disulfide patterns, i.e., the cysteine connectivity, is crucial to achieve a global picture of the active conformation of the protein of interest. Electron-transfer dissociation (ETD) constitutes a valuable tool to cleave the disulfide bonds in the gas phase, avoiding chemical reduction/alkylation in solution. To characterize the cysteine pairing, the present work proposes (i) to reduce by ETD one of the two disulfide bridges of model peptides, resulting in the opening of the cyclic structures, (ii) to separate the generated species by ion mobility, and (iii) to characterize the species using collision-induced dissociation (CID). Results of this strategy applied to several peptides show different behaviors depending on the connectivity. The loss of SH· radical species, observed for all the peptides, confirms the cleavage of the disulfides during the ETD process.