The Strategy for Peptidomic LC-MS/MS Data Analysis: The Case of Urinary Peptidome Study.

The study of urinary peptidome is an important area of research, which concerns the characterization of endogenous peptides, as well as the identification of biomarkers for a wide range of socially significant diseases. First of all, this relates to renal and genitourinary pathologies and/or pathologies associated with proteinuria, such as kidney diseases, bladder, prostate and ovarian cancers, diabetic nephropathy, and pre-eclampsia. Unlike proteins, peptides do not require proteolytic hydrolysis, can be analyzed in their native form and can provide certain information about occurring (patho)physiological processes. Mass spectrometry (MS)-based approaches are the most unbiased and sensitive instruments with high multiplexing capacity and provided most of the current information about endogenous urine peptides. However, despite the large number of urine peptidomic studies, there are certain issues related to the insufficient comparability of their results due to the lack of consistent approaches to their interpretation. Also the development of a custom project-specific protein library for endogenous peptides search and identification is another important point that should be noted in the context of high-throughput peptidomic analysis. Here we propose the custom-specific urinary protein database and the grouping of endogenous urinary peptides with overlapping sequences as useful tools, which can facilitate the acquisition and analysis of LC-MS peptidomic data, as well as the comparison of results of different studies, which should facilitate their more efficient further application.

Targeted MRM Quantification of Urinary Proteins in Chronic Kidney Disease Caused by Glomerulopathies.

Glomerulopathies with nephrotic syndrome that are resistant to therapy often progress to end-stage chronic kidney disease (CKD) and require timely and accurate diagnosis. Targeted quantitative urine proteome analysis by mass spectrometry (MS) with multiple-reaction monitoring (MRM) is a promising tool for early CKD diagnostics that could replace the invasive biopsy procedure. However, there are few studies regarding the development of highly multiplexed MRM assays for urine proteome analysis, and the two MRM assays for urine proteomics described so far demonstrate very low consistency. Thus, the further development of targeted urine proteome assays for CKD is actual task. Herein, a BAK270 MRM assay previously validated for blood plasma protein analysis was adapted for urine-targeted proteomics. Because proteinuria associated with renal impairment is usually associated with an increased diversity of plasma proteins being present in urine, the use of this panel was appropriate. Another advantage of the BAK270 MRM assay is that it includes 35 potential CKD markers described previously. Targeted LC-MRM MS analysis was performed for 69 urine samples from 46 CKD patients and 23 healthy controls, revealing 138 proteins that were found in ≥2/3 of the samples from at least one of the groups. The results obtained confirm 31 previously proposed CKD markers. Combination of MRM analysis with machine learning for data processing was performed. As a result, a highly accurate classifier was developed (AUC = 0.99) that enables distinguishing between mild and severe glomerulopathies based on the assessment of only three urine proteins (GPX3, PLMN, and A1AT or SHBG).

In Vitro Analysis of Biological Activity of Circulating Cell-Free DNA Isolated from Blood Plasma of Schizophrenic Patients and Healthy Controls-Part 2: Adaptive Response.

Oxidized in vitro genomic DNA (gDNA) is known to launch an adaptive response in human cell cultures. The cfDNA extracted from the plasma of schizophrenic patients (sz-cfDNA) and healthy controls (hc-cfDNA) contains increased amounts of 8-oxodG, a DNA-oxidation marker. The aim of the research was answering a question: can the human cfDNA isolated from blood plasma stimulate the adaptive response in human cells? In vitro responses of ten human skin fibroblasts (HSFs) and four peripheral blood mononuclear cell (PBMC) lines after 1-24 h of incubation with sz-cfDNA, gDNA and hc-cfDNA containing different amounts of 8-oxodG were examined. Expressions of RNA of eight genes (NOX4, NFE2L2, SOD1, HIF1A, BRCA1, BRCA2, BAX and BCL2), six proteins (NOX4, NRF2, SOD1, HIF1A, γH2AX and BRCA1) and DNA-oxidation marker 8-oxodG were analyzed by RT-qPCR and flow cytometry (when analyzing the data, a subpopulation of lymphocytes (PBL) was identified). Adding hc-cfDNA or sz-cfDNA to HSFs or PBMC media in equal amounts (50 ng/mL, 1-3 h) stimulated transient synthesis of free radicals (ROS), which correlated with an increase in the expressions of NOX4 and SOD1 genes and with an increase in the levels of the markers of DNA damage γH2AX and 8-oxodG. ROS and DNA damage induced an antioxidant response (expression of NFE2L2 and HIF1A), DNA damage response (BRCA1 and BRCA2 gene expression) and anti-apoptotic response (changes in BAX and BCL2 genes expression). Heterogeneity of cells of the same HSFs or PBL population was found with respect to the type of response to (sz,hc)-cfDNA. Most cells responded to oxidative stress with an increase in the amount of NRF2 and BRCA1 proteins along with a moderate increase in the amount of NOX4 protein and a low amount of 8-oxodG oxidation marker. However, upon the exposure to (sz,hc)-cfDNA, the size of the subpopulation with apoptosis signs (high DNA damage degree, high NOX4 and low NRF2 and BRCA1 levels) also increased. No significant difference between the responses to sz-cfDNA and hc-cfDNA was observed. Sz-cfDNA and hc-cfDNA showed similarly high bioactivity towards fibroblasts and lymphocytes. Conclusion: In cultured human cells, hc-cfDNA and sz-cfDNA equally stimulated an adaptive response aimed at launching the antioxidant, repair, and anti-apoptotic processes. The mediator of the development of the adaptive response are ROS produced by, among others, NOX4 and SOD1 enzymes.

Proteomic Markers and Early Prediction of Alzheimer's Disease.

Alzheimer's disease (AD) is the most common socially significant neurodegenerative pathology, which currently affects more than 30 million elderly people worldwide. Since the number of patients grows every year and may exceed 115 million by 2050, and due to the lack of effective therapies, early prediction of AD remains a global challenge, solution of which can contribute to the timely appointment of a preventive therapy in order to avoid irreversible changes in the brain. To date, clinical assays for the markers of amyloidosis in cerebrospinal fluid (CSF) have been developed, which, in conjunction with the brain MRI and PET studies, are used either to confirm the diagnosis based on obligate clinical criteria or to predict the risk of AD developing at the stage of mild cognitive impairment (MCI). However, the problem of predicting AD at the asymptomatic stage remains unresolved. In this regard, the search for new protein markers and studies of proteomic changes in CSF and blood plasma are of particular interest and may consequentially identify particular pathways involved in the pathogenesis of AD. Studies of specific proteomic changes in blood plasma deserve special attention and are of increasing interest due to the much less invasive method of sample collection as compared to CSF, which is important when choosing the object for large-scale screening. This review briefly summarizes the current knowledge on proteomic markers of AD and considers the prospects of developing reliable methods for early identification of AD risk factors based on the proteomic profile.

NADPH-oxidase 4 gene over-expression in peripheral blood lymphocytes of the schizophrenia patients.

INTRODUCTION: Increased systemic oxidative stress is common in schizophrenia (SZ) patients. NADPH-oxidase 4 (NOX4) is the cell oxidoreductase, catalyzing the hydrogen peroxide formation. Presumably, NOX4 is the main oxidative stress factor in a number of diseases such as cardiovascular diseases and cancer. We hypothesized that NOX4 may be involved in the oxidative stress development caused by the disease in the schizophrenic patients' peripheral blood lymphocytes (PBL). MATERIALS AND METHODS: The SZ group included 100 patients (68 men and 32 women aged 28 ± 11 years). The control group included 60 volunteers (35 men and 25 women aged 25 ± 12 years). Flow cytometry analysis (FCA) was used for DNA damage markers (8-oxodG, É£H2AX), pro- and antiapoptotic proteins (BAX1 and BCL2) and the master-regulator of anti-oxidant response NRF2 detection in the lymphocytes of the untreated SZ patients (N = 100) and the healthy control (HC, N = 60). FCA and RT-qPCR were used for NOX4 and RNANOX4 detection in the lymphocytes. RT-qPCR was used for mtDNA quantitation in peripheral blood mononuclear cells. Cell-free DNA concentration was determined in blood plasma fluorimetrically. RESULTS: 8-oxodG, NOX4, and BCL2 levels in the PBL in the SZ group were higher than those in the HC group (p < 0.001). É£H2AX protein level was increased in the subgroup with high 8-oxodG (p<0.02) levels and decreased in the subgroup with low 8-oxodG (p <0.0001) levels. A positive correlation was found between 8-oxodG, É£H2AX and BAX1 levels in the SZ group (p <10-6). NOX4 level in lymphocytes did not depend on the DNA damage markers values and BAX1 and BCL2 proteins levels. In 15% of PBL of the HC group a small cellular subfraction was found (5-12% of the total lymphocyte pool) with high DNA damage level and elevated BAX1 protein level. The number of such cells was maximal in PBL samples with low NOX4 protein levels. CONCLUSION: Significant NOX4 gene expression was found a in SZ patients' lymphocytes, but the corresponding protein is probably not a cause of the DNA damage.

Urinary Protein and Peptide Markers in Chronic Kidney Disease.

Chronic kidney disease (CKD) is a non-specific type of kidney disease that causes a gradual decline in kidney function (from months to years). CKD is a significant risk factor for death, cardiovascular disease, and end-stage renal disease. CKDs of different origins may have the same clinical and laboratory manifestations but different progression rates, which requires early diagnosis to determine. This review focuses on protein/peptide biomarkers of the leading causes of CKD: diabetic nephropathy, IgA nephropathy, lupus nephritis, focal segmental glomerulosclerosis, and membranous nephropathy. Mass spectrometry (MS) approaches provided the most information about urinary peptide and protein contents in different nephropathies. New analytical approaches allow urinary proteomic-peptide profiles to be used as early non-invasive diagnostic tools for specific morphological forms of kidney disease and may become a safe alternative to renal biopsy. MS studies of the key pathogenetic mechanisms of renal disease progression may also contribute to developing new approaches for targeted therapy.

The Dynamics of ß-Amyloid Proteoforms Accumulation in the Brain of a 5xFAD Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is the leading cause of dementia among the elderly. Neuropathologically, AD is characterized by the deposition of a 39- to 42-amino acid long ß-amyloid (Aß) peptide in the form of senile plaques. Several post-translational modifications (PTMs) in the N-terminal domain have been shown to increase the aggregation and cytotoxicity of Aß, and specific Aß proteoforms (e.g., Aß with isomerized D7 (isoD7-Aß)) are abundant in the senile plaques of AD patients. Animal models are indispensable tools for the study of disease pathogenesis, as well as preclinical testing. In the presented work, the accumulation dynamics of Aß proteoforms in the brain of one of the most widely used amyloid-based mouse models (the 5xFAD line) was monitored. Mass spectrometry (MS) approaches, based on ion mobility separation and the characteristic fragment ion formation, were applied. The results indicated a gradual increase in the Aß fraction of isoD7-Aß, starting from approximately 8% at 7 months to approximately 30% by 23 months of age. Other specific PTMs, in particular, pyroglutamylation, deamidation, and oxidation, as well as phosphorylation, were also monitored. The results for mice of different ages demonstrated that the accumulation of Aß proteoforms correlate with the formation of Aß deposits. Although the mouse model cannot be a complete analogue of the processes occurring in the human brain in AD, and several of the observed parameters differ significantly from human values supposedly due to the limited lifespan of the model animals, this dynamic study provides evidence on at least one of the possible mechanisms that can trigger amyloidosis in AD, i.e., the hypothesis on the relationship between the accumulation of isoD7-Aß and the progression of AD-like pathology.

Two types of procoagulant platelets are formed upon physiological activation and are controlled by integrin α(IIb)ß(3).

OBJECTIVE: Phosphatidylserine (PS) externalization by platelets upon activation is a key event in hemostasis and thrombosis. It is currently believed that strong stimulation of platelets forms 2 subpopulations, only 1 of which expresses PS. METHODS AND RESULTS: Here, we demonstrate that physiological stimulation leads to the formation of not 1 but 2 types of PS-expressing activated platelets, with dramatically different properties. One subpopulation sustained increased calcium level after activation, whereas another returned to the basal low-calcium state. High-calcium PS-positive platelets had smaller size, high surface density of fibrin(ogen), no active integrin α(IIb)ß(3), depolarized mitochondrial membranes, gradually lost cytoplasmic membrane integrity, and were poorly aggregated. In contrast, the low-calcium PS-positive platelets had normal size, retained mitochondrial membrane potential and cytoplasmic membrane integrity, and combined retention of fibrin(ogen) with active α(IIb)ß(3) and high proaggregatory function. Formation of low-calcium PS-positive platelets was promoted by platelet concentration increase or shaking and was decreased by integrin α(IIb)ß(3) antagonists, platelet dilution, or in platelets from kindlin-3-deficient and Glanzmann thrombasthenia patients. CONCLUSIONS: Identification of a novel PS-expressing platelet subpopulation with low calcium regulated by integrin α(IIb)ß(3) can be important for understanding the mechanisms of PS exposure and thrombus formation.