Academic literature on the topic 'Cysteinylated albumin'

Exposure of blood plasma/serum (P/S) to thawed conditions (> −30 °C) can produce biomolecular changes that skew measurements of biomarkers within archived patient samples, potentially rendering them unfit for molecular analysis. Because freeze-thaw histories are often poorly documented, objective methods for assessing molecular fitness before analysis are needed. We report a 10-μl, dilute-and-shoot, intact-protein mass spectrometric assay of albumin proteoforms called “ΔS-Cys-Albumin” that quantifies cumulative exposure of archived P/S samples to thawed conditions. The relative abundance of S-cysteinylated (oxidized) albumin in P/S increases inexorably but to a maximum value under 100% when samples are exposed to temperatures > −30 °C. The difference in the relative abundance of S-cysteinylated albumin (S-Cys-Alb) before and after an intentional incubation period that drives this proteoform to its maximum level is denoted as ΔS-Cys-Albumin. ΔS-Cys-Albumin in fully expired samples is zero. The range (mean ± 95% CI) observed for ΔS-Cys-Albumin in fresh cardiac patient P/S (n = 97) was, for plasma 12–29% (20.9 ± 0.75%) and for serum 10–24% (15.5 ± 0.64%). The multireaction rate law that governs S-Cys-Alb formation in P/S was determined and shown to predict the rate of formation of S-Cys-Alb in plasma and serum samples—a step that enables back-calculation of the time at which unknown P/S specimens have been exposed to room temperature. A blind challenge demonstrated that ΔS-Cys-Albumin can detect exposure of groups (n = 6 each) of P/S samples to 23 °C for 2 h, 4 °C for 16 h, or −20 °C for 24 h—and exposure of individual specimens for modestly increased times. An unplanned case study of nominally pristine serum samples collected under NIH-sponsorship demonstrated that empirical evidence is required to ensure accurate knowledge of archived P/S biospecimen storage history.

In the present paper, the extracellular antioxidant activity of N-acetyl-cysteine (NAC) is explained by considering its ability to regenerate the free form of albumin Cys34 by breaking the disulfide bond of the cysteinylated form (HSA-Cys). NAC’s capability to regenerate albumin Cys34 (HSA-SH) was studied by MS intact protein analysis in human plasma and in a concentration range of NAC easily achievable after oral and i.v. administration (5–50 µg/mL). NAC dose-dependently broke the HSA-Cys bond to form the dimer NAC-Cys thus regenerating Cys34, whose reduced state was maintained for at least 120 min. Cys was faster in restoring Cys34, according to the reaction constant determined with the glutathione disulfide (GSSG) reaction, but after 60 min the mixed disulfide HSA-Cys turned back due to the reaction of the dimer Cys-Cys with Cys34. The explanation for the different rate exchanges between Cys-Cys and Cys-NAC with Cys34 was given by molecular modeling studies. Finally, the Cys34 regenerating effect of NAC was related to its ability to improve the total antioxidant capacity of plasma (TRAP assay). The results well indicate that NAC greatly increases the plasma antioxidant activity and this effect is not reached by a direct effect but through the regenerating effect of Cys34.

We determined albumin post-translational modifications (PTMs) by mass spectrometry (MS) in plasma and cerebrospinal fluid (CSF) from 31 Alzheimer’s disease (AD) patients (with 27 samples of paired plasma-CSF from the same patients). Results were cross-sectionally compared with healthy controls. For percentage of relative intensity of glycated isoforms, plasma albumin was globally more glycated in AD patients than in healthy controls (P<0.01). MS results in plasma were confirmed by a quantitative enzymatic assay (Lucica GA-L) for albumin early-glycation detection. In CSF there were no global glycation differences detected by MS, although a different pattern of glycated isoforms was observed. Oxidized+glycated and cysteinylated+glycated isoforms were increased in both plasma and CSF of AD patients in comparison with healthy controls (P<0.001). Furthermore, AD patients showed higher glycation in plasma than in CSF (P<0.01). Our data support the role of glycation and oxidative stress in AD.

Increasing body of evidence supports that oxidative stress is involved systolic and diastolic myocardial dysfunction in HF. Indeed, it has been well established an association between cardiac remodeling (hypertrophy, apoptosis or contractile dysfunction) and oxidative stress. Hence, the identification, quantification and fully characterization of oxidized biomolecules present in the plasma of HF patients can contribute to understand the mechanisms responsible for disease development but also as biological markers. However, the classic biomarkers of oxidative stress identified in HF do not provide thorough information but report about a general oxidative stress status. For a better understanding of the pathology of HF it is needed to unveil not only protein and lipid targets of oxidative damage but also the residues undergoing the modification within the protein and the structure of the oxidation products. Analysis of all of these oxPTMs and lipid peroxidation products by mass spectrometry is extremely challenging. Indeed, oxPTMs tend to occur randomly on a number of susceptible residues and proteins, making it extremely difficult to define all protein species. Along this project, plasma samples from HF patients belonging to different NYHA groups (from class I to class IV) have been employed to identify oxidized biomolecules as potential biomarkers of HF, and eventually subjected to several plasma prefractionation strategies combined with high-throughput untargeted and targeted mass spectrometry techniques. Consequently, part of this project has been focused on the study of oxidative modifications in human serum albumin (HSA), the most abundant protein in the circulatory system, associated with HF. HSA is involved in a wide range of biological functions and, due to its long half-life and high concentration in plasma, HSA is highly sensitive to undergo oxPTMs that may lead to its functional loss, thus contributing to the progression of HF. Taking advantage of the high abundance of HSA, we relatively quantified for the first time plasma levels of cysteinylated HSA (cys-HSA) and also levels of early glycated HSA (GA), and we observed a significant increase in HF patients with respect to the healthy subjects. A positive correlation between the levels of both isoforms and HF severity was highlighted whereas the abundance of total HSA showed a tendency to decrease when raising the severity degree of HF. For a deeper characterization of GA, we elucidated for the first time the early glycation pattern of HSA associated to HF by means of the newest generation of Tribrid MS instruments. Lys233 and lys525 were observed as the two most abundant glycated amino acids (79% and 13% respectively). Considering that further modifications of GA, such as rearrangement, oxidation, polymerization, and cleavage give rise to irreversible conjugates, called advanced glycation end products (AGEs), we also aimed to unveil the plasma advanced glycation pattern associated to HF. In this case, lys20, arg98, and lys402 emerged as the three most abundant carboxymethylated residues. Therefore, the results suggested that on one hand lys233 and lys525, and on the other lys20, arg98 and lys402, might represent the main potential therapeutic targets to reduce GA and AGE-HSA levels in HF patients, respectively. Besides the study of oxidized isoforms of HSA, we also tried to identify other less abundant advanced glycation and lipoxidation end products (AGEs and ALEs) in plasma by means of an enrichment strategy based on the ability of RAGE receptor to bind AGEs and ALEs. Due to the relevance of HSA in circulation, we have also evaluated the potential causal role of GA in the etiopathogenesis of HF. Hence, we pursued to evaluate the biological effects of GA on cardiac myocytes. Results highlighted that GA modulates in vitro the cardiomyocyte expression of inflammatory cytokines such as IL-6 or TNF-α. Furthermore, we reported that GA induces oxidative stress and oxidative modifications of a multitude of cellular proteins within cardiac myocytes. In addition, GA modulates the secretion of several cardiac proteins involved in response to stress biological processes. On the other hand, lipids play a crucial role in physiological processes and phospholipid disruption may participate in cardiovascular disease events, therefore the phospholipidome profiling has emerged as a powerful tool to explore novel biomarkers and mechanisms in several pathologies. Hence, we have also studied for the first time the plasma phospholipidome of HF patients. In this pilot study, we have applied both untargeted and targeted (MRM) strategies together with fractionating methods to separate the phospholipid content from the rest of plasma components, in order to decrease the heterogeneity and complexity of this biological matrix. The results suggested that HF plasma phospholipid signatures were gender-specific. In the case of females, several PE species were more abundant in HF than in the control group. On the contrary, male HF patients mainly showed a higher abundance of LPC species when compared to the male control group. Additionally, lipidomic approaches (LC-MS/MS and PRM) have been carried out to define for the first time a circulating oxidative lipid pattern associated with HF. In this case, we have focused on long- and short-chain products of lipid peroxidation since their identification and characterization in the pathology of HF were still lacking. Promising results have been pinpointed so far: we have detected 9 lipid peroxidation products (full-chain oxidized lipids together with fragmented species). In conclusion, the results gathered along this Ph.D. project provide evidence at the molecular level of the mechanisms associated to oxidative stress and oxidative damage underlying the development and progression of HF, thus contributing to a better understanding of the pathology. Indeed, the elucidation of specific modified residues within key circulating proteins such as HSA, or the characterization of long- and fragmented-chain lipid peroxidation products present in HF plasma samples reported in this thesis may pave the way to new therapies to reduce and treat HF. We have also contributed to expand the awareness of the biological effects of GA in the cardiac context. Additionally, this project has pointed out a gender-dependent adaptation of the plasma phospholipidome occurring in the pathology of HF, highlighting that gender is a major and often underestimated factor that should be carefully considered when screening lipidomes or proteomes of pathological processes. Finally, we have demonstrated that either proteomic or lipidomic technologies can provide deep biological insight into human health and disease.

abstract: Exposure of blood plasma/serum (P/S) to thawed conditions, greater than -30°C, can produce biomolecular changes that misleadingly impact measurements of clinical markers within archived samples. Reported here is a low sample-volume, dilute-and-shoot, intact protein mass spectrometric assay of albumin proteoforms called “ΔS-Cys-Albumin” that quantifies cumulative exposure of archived P/S samples to thawed conditions. The assay uses the fact that S-cysteinylation (oxidation) of albumin in P/S increases to a maximum value when exposed to temperatures greater than -30°C. The multi-reaction rate law that governs this albumin S-cysteinylation formation in P/S was determined and was shown to predict the rate of formation of S-cysteinylated albumin in P/S samples—a step that enables back-calculation of the time at which unknown P/S specimens have been exposed to room temperature. To emphasize the capability of this assay, a blind challenge demonstrated the ability of ΔS-Cys-Albumin to detect exposure of individual and grouped P/S samples to unfavorable storage conditions. The assay was also capable of detecting an anomaly in a case study of nominally pristine serum samples collected under NIH-sponsorship, demonstrating that empirical evidence is required to guarantee accurate knowledge of archived P/S biospecimen storage history. The ex vivo glycation of human serum albumin was also investigated showing that P/S samples stored above their freezing point leads to significant increases in glycated albumin. These increases were found to occur within hours at room temperature, and within days at -20 °C. These increases continued over a period of 1-2 weeks at room temperature and over 200 days at -20 °C, ultimately resulting in a doubling of glycated albumin in both healthy and diabetic patients. It was also shown that samples stored at lower surface area-to-volume ratios or incubated under a nitrogen atmosphere experienced less rapid glucose adduction of albumin—suggesting a role for oxidative glycation in the ex vivo glycation of albumin.
Dissertation/Thesis
Doctoral Dissertation Biochemistry 2018