Targeted MRM-analysis of plasma proteins in frozen whole blood samples from patients with COVID-19: a retrospective study.

OBJECTIVES: The COVID-19 pandemic has exposed a number of key challenges that need to be urgently addressed. Mass spectrometric studies of blood plasma proteomics provide a deep understanding of the relationship between the severe course of infection and activation of specific pathophysiological pathways. Analysis of plasma proteins in whole blood may also be relevant for the pandemic as it requires minimal sample preparation. METHODS: The frozen whole blood samples were used to analyze 203 plasma proteins using multiple reaction monitoring (MRM) mass spectrometry and stable isotope-labeled peptide standards (SIS). A total of 131 samples (FRCC, Russia) from patients with mild (n=41), moderate (n=39) and severe (n=19) COVID-19 infection and healthy controls (n=32) were analyzed. RESULTS: Levels of 94 proteins were quantified and compared. Significant differences between all of the groups were revealed for 44 proteins. Changes in the levels of 61 reproducible COVID-19 markers (SERPINA3, SERPING1, ORM1, HRG, LBP, APOA1, AHSG, AFM, ITIH2, etc.) were consistent with studies performed with serum/plasma samples. The best-performing classifier built with 10 proteins achieved the best combination of ROC-AUC (0.97-0.98) and accuracy (0.90-0.93) metrics and distinguished patients from controls, as well as patients by severity. CONCLUSIONS: Here, for the first time, frozen whole blood samples were used for proteomic analysis and assessment of the status of patients with COVID-19. The results obtained with frozen whole blood samples are consistent with those from plasma and serum.

Mass spectrometric studies of the variety of beta-amyloid proteoforms in Alzheimer's disease.

This review covers the results of the application of mass spectrometric (MS) techniques to study the diversity of beta-amyloid (Aß) peptides in human samples. Since Aß is an important hallmark of Alzheimer's disease (AD), which is a socially significant neurodegenerative disorder of the elderly worldwide, analysis of its endogenous variations is of particular importance for elucidating the pathogenesis of AD, predicting increased risks of the disease onset, and developing effective therapy. MS approaches have no alternative for the study of complex samples, including a wide variety of Aß proteoforms, differing in length and modifications. Approaches based on matrix-assisted laser desorption/ionization time-of-flight and liquid chromatography with electrospray ionization tandem MS are most common in Aß studies. However, Aß forms with isomerized and/or racemized Asp and Ser residues require the use of special methods for separation and extra sensitive and selective methods for detection. Overall, this review summarizes current knowledge of Aß species found in human brain, cerebrospinal fluid, and blood plasma; focuses on application of different MS approaches for Aß studies; and considers the potential of MS techniques for further studies of Aß-peptides.

Molecular Mechanism of Zinc-Dependent Oligomerization of Alzheimer's Amyloid-ß with Taiwan (D7H) Mutation.

Amyloid-ß (Aß) is a peptide formed by 39-43 amino acids, heterogenous by the length of its C-terminus. Aß constitutes a subnanomolar monomeric component of human biological fluids; however, in sporadic variants of Alzheimer's disease (AD), it forms soluble neurotoxic oligomers and accumulates as insoluble extracellular polymeric aggregates (amyloid plaques) in the brain tissues. The plaque formation is controlled by zinc ions; therefore, abnormal interactions between the ions and Aß seem to take part in the triggering of sporadic AD. The amyloid plaques contain various Aß isoforms, among which the most common is Aß with an isoaspartate in position 7 (isoD7). The spontaneous conversion of D7 to isoD7 is associated with Aß aging. Aß molecules with isoD7 (isoD7-Aß) easily undergo zinc-dependent oligomerization, and upon administration to transgenic animals (mice, nematodes) used for AD modeling, act as zinc-dependent seeds of the pathological aggregation of Aß. The formation of zinc-bound homo- and hetero-oligomers with the participation of isoD7-Aß is based on the rigidly structured segment 11-EVHH-14, located in the Aß metal binding domain (Aß16). Some hereditary variants of AD are associated with familial mutations within the domain. Among these, the most susceptible to zinc-dependent oligomerization is Aß with Taiwan (D7H) mutation (D7H-Aß). In this study, the D7H-Aß metal binding domain (D7H-Aß16) has been used as a model to establish the molecular mechanism of zinc-induced D7H-Aß oligomerization through turbidimetry, dynamic light scattering, isothermal titration calorimetry, mass spectrometry, and computer modelling. Additionally, the modeling data showed that a molecule of D7H-Aß, as well as isoD7-Aß in combination with two Aß molecules, renders a stable zinc-induced heterotrimer. The trimers are held together by intermolecular interfaces via zinc ions, with the primary interfaces formed by 11-EVHH-14 sites of the interacting trimer subunits. In summary, the obtained results confirm the role of the 11-EVHH-14 region as a structure and function determinant for the zinc-dependent oligomerization of all known Aß species (including various chemically modified isoforms and AD-associated mutants) and point at this region as a potent target for drugs aimed to stop amyloid plaque formation in both sporadic and hereditary variants of AD.

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).

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.

Potential Urine Proteomic Biomarkers for Focal Segmental Glomerulosclerosis and Minimal Change Disease.

Primary focal segmental glomerulosclerosis (FSGS), along with minimal change disease (MCD), are diseases with primary podocyte damage that are clinically manifested by the nephrotic syndrome. The pathogenesis of these podocytopathies is still unknown, and therefore, the search for biomarkers of these diseases is ongoing. Our aim was to determine of the proteomic profile of urine from patients with FSGS and MCD. Patients with a confirmed diagnosis of FSGS (n = 30) and MCD (n = 9) were recruited for the study. For a comprehensive assessment of the severity of FSGS a special index was introduced, which was calculated as follows: the first score was assigned depending on the level of eGFR, the second score-depending on the proteinuria level, the third score-resistance to steroid therapy. Patients with the sum of these scores of less than 3 were included in group 1, with 3 or more-in group 2. The urinary proteome was analyzed using liquid chromatography/mass spectrometry. The proteome profiles of patients with severe progressive FSGS from group 2, mild FSGS from group 1 and MCD were compared. Results of the label free analysis were validated using targeted LC-MS based on multiple reaction monitoring (MRM) with stable isotope labelled peptide standards (SIS) available for 47 of the 76 proteins identified as differentiating between at least one pair of groups. Quantitative MRM SIS validation measurements for these 47 proteins revealed 22 proteins with significant differences between at least one of the two group pairs and 14 proteins were validated for both comparisons. In addition, all of the 22 proteins validated by MRM SIS analysis showed the same direction of change as at the discovery stage with label-free LC-MS analysis, i.e., up or down regulation in MCD and FSGS1 against FSGS2. Patients from the FSGS group 2 showed a significantly different profile from both FSGS group 1 and MCD. Among the 47 significantly differentiating proteins, the most significant were apolipoprotein A-IV, hemopexin, vitronectin, gelsolin, components of the complement system (C4b, factors B and I), retinol- and vitamin D-binding proteins. Patients with mild form of FSGS and MCD showed lower levels of Cystatin C, gelsolin and complement factor I.

Prognosis of Alzheimer's Disease Using Quantitative Mass Spectrometry of Human Blood Plasma Proteins and Machine Learning.

Early recognition of the risk of Alzheimer's disease (AD) onset is a global challenge that requires the development of reliable and affordable screening methods for wide-scale application. Proteomic studies of blood plasma are of particular relevance; however, the currently proposed differentiating markers are poorly consistent. The targeted quantitative multiple reaction monitoring (MRM) assay of the reported candidate biomarkers (CBs) can contribute to the creation of a consistent marker panel. An MRM-MS analysis of 149 nondepleted EDTA-plasma samples (MHRC, Russia) of patients with AD (n = 47), mild cognitive impairment (MCI, n = 36), vascular dementia (n = 8), frontotemporal dementia (n = 15), and an elderly control group (n = 43) was performed using the BAK 125 kit (MRM Proteomics Inc., Canada). Statistical analysis revealed a significant decrease in the levels of afamin, apolipoprotein E, biotinidase, and serum paraoxonase/arylesterase 1 associated with AD. Different training algorithms for machine learning were performed to identify the protein panels and build corresponding classifiers for the AD prognosis. Machine learning revealed 31 proteins that are important for AD differentiation and mostly include reported earlier CBs. The best-performing classifiers reached 80% accuracy, 79.4% sensitivity and 83.6% specificity and were able to assess the risk of developing AD over the next 3 years for patients with MCI. Overall, this study demonstrates the high potential of the MRM approach combined with machine learning to confirm the significance of previously identified CBs and to propose consistent protein marker panels.

Quantitative Assessment of Serine-8 Phosphorylated ß-Amyloid Using MALDI-TOF Mass Spectrometry.

The study of the molecular mechanisms of the pathogenesis of Alzheimer's disease (AD) is extremely important for identifying potential therapeutic targets as well as early markers. In this regard, the study of the role of post-translational modifications (PTMs) of ß-amyloid (Aß) peptides is of particular relevance. Serine-8 phosphorylated forms (pSer8-Aß) have been shown to have an increased aggregation capacity and may reflect the severity of amyloidosis. Here, an approach for quantitative assessment of pSer8-Aß based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is proposed. The relative fraction of pSer8-Aß was estimated in the total Aß-pool with a detection limit of 1 fmol for pSer8-Aß (1-16) and an accuracy of 2% for measurements in the reflectron mode. The sensitivity of the developed method is suitable for determining the proportion of phosphorylated peptides in biological samples.

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.

Conservative and Atypical Ferritins of Sponges.

Ferritins comprise a conservative family of proteins found in all species and play an essential role in resistance to redox stress, immune response, and cell differentiation. Sponges (Porifera) are the oldest Metazoa that show unique plasticity and regenerative potential. Here, we characterize the ferritins of two cold-water sponges using proteomics, spectral microscopy, and bioinformatic analysis. The recently duplicated conservative HdF1a/b and atypical HdF2 genes were found in the Halisarca dujardini genome. Multiple related transcripts of HpF1 were identified in the Halichondria panicea transcriptome. Expression of HdF1a/b was much higher than that of HdF2 in all annual seasons and regulated differently during the sponge dissociation/reaggregation. The presence of the MRE and HRE motifs in the HdF1 and HdF2 promotor regions and the IRE motif in mRNAs of HdF1 and HpF indicates that sponge ferritins expression depends on the cellular iron and oxygen levels. The gel electrophoresis combined with specific staining and mass spectrometry confirmed the presence of ferric ions and ferritins in multi-subunit complexes. The 3D modeling predicts the iron-binding capacity of HdF1 and HpF1 at the ferroxidase center and the absence of iron-binding in atypical HdF2. Interestingly, atypical ferritins lacking iron-binding capacity were found in genomes of many invertebrate species. Their function deserves further research.

Mass-Spectrometric Detection of SARS-CoV-2 Virus in Scrapings of the Epithelium of the Nasopharynx of Infected Patients via Nucleocapsid N Protein.

The detection of viral RNA by polymerase chain reaction (PCR) is currently the main diagnostic tool for COVID-19 ( Eurosurveillance 2019, 25 (3), 1). The PCR-based test, however, shows limited sensitivity, especially in the early and late stages of disease development ( Nature 2020, 581, 465-469; J. Formosan Med. Assoc. 2020, 119 (6) 1123), and is relatively time-consuming. Fast and reliable complementary methods for detecting the viral infection would be of help in the current pandemic conditions. Mass spectrometry is one of such possibilities. We have developed a mass-spectrometry-based method for the detection of the SARS CoV-2 virus in nasopharynx epithelial swabs based on the detection of the viral nucleocapsid N protein. Our approach shows confident identification of the N protein in patient samples, even those with the lowest viral loads, and a much simpler preparation procedure. Our main protocol consists of virus inactivation by heating and the addition of isopropanol and tryptic digestion of the proteins sedimented from the swabs followed by MS analysis. A set of unique peptides, produced as a result of proteolysis of the nucleocapsid phosphoprotein of SARS-CoV-2, is detected. The obtained results can further be used to create fast parallel mass-spectrometric approaches for the detection of the virus in the nasopharyngeal mucosa, saliva, sputum and other physiological fluids.

Probabilistic model applied to ion abundances in product-ion spectra: quantitative analysis of aspartic acid isomerization in peptides.

Evaluation of post-translational modifications of protein molecules is important for both basic and applied biomedical research. Mass spectrometric quantitative studies of modifications, which do not change the mass of the protein, such as isomerization of aspartic acid, do not necessarily require the use of isotope-labelled standards. However, the accurate solution of this problem requires a deep understanding of the relationship between the mole fractions of the isomers and the peak intensities in the mass spectra. In previous studies on the isomerization of aspartic acid in short beta-amyloid fragments, it has been shown that calibration curves used for such quantitative studies often have a non-linear form. The reason for the deviation in the shape of the calibration curves from linearity has not yet been established. Here, we propose an explanation for this phenomenon based on a probabilistic model of the fragmentation process and present a general approach for the selection of fragments that can be used for quantitative studies of the degree of isomerization. Graphical Abstract.

The Effects of Spaceflight Factors on the Human Plasma Proteome, Including Both Real Space Missions and Ground-Based Experiments.

The aim of the study was to compare proteomic data on the effects of spaceflight factors on the human body, including both real space missions and ground-based experiments. LC-MS/MS-based proteomic analysis of blood plasma samples obtained from 13 cosmonauts before and after long-duration (169-199 days) missions on the International Space Station (ISS) and for five healthy men included in 21-day-long head-down bed rest (HDBR) and dry immersion experiments were performed. The semi-quantitative label-free analysis revealed significantly changed proteins: 19 proteins were significantly different on the first (+1) day after landing with respect to background levels; 44 proteins significantly changed during HDBR and 31 changed in the dry immersion experiment. Comparative analysis revealed nine common proteins (A1BG, A2M, SERPINA1, SERPINA3, SERPING1, SERPINC1, HP, CFB, TF), which changed their levels after landing, as well as in both ground-based experiments. Common processes, such as platelet degranulation, hemostasis, post-translational protein phosphorylation and processes of protein metabolism, indicate common pathogenesis in ground experiments and during spaceflight. Dissimilarity in the lists of significantly changed proteins could be explained by the differences in the dynamics of effective development in the ground-based experiments. Data are available via ProteomeXchange using the identifier PXD013305.

A Binuclear Zinc Interaction Fold Discovered in the Homodimer of Alzheimer's Amyloid-ß Fragment with Taiwanese Mutation D7H.

Zinc-induced oligomerization of amyloid-ß peptide (Aß) produces potentially pathogenic agents of Alzheimer's disease. Mutations and modifications in the metal binding domain 1-16 of Aß peptide crucially affect its zinc-induced oligomerization by changing intermolecular zinc mediated interface. The 3D structure of this interface appearing in a range of Aß species is a prospective drug target for disease modifying therapy. Using NMR spectroscopy, EXAFS spectroscopy, mass spectrometry, and isothermal titration calorimetry the interaction of zinc ions with Aß fragments 1-7 and 1-10 carrying familial Taiwanese mutation D7H was studied. Zinc ions induce formation of a stable homodimer formed by the two peptide chains fastened by two zinc ions and stacking interactions of imidazole rings. A binuclear zinc interaction fold in the dimer structure was discovered. It can be used for designing zinc-regulated proteins and zinc-mediated self-assembling peptides.

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.

Absolute Quantitative Targeted Monitoring of Potential Plasma Protein Biomarkers: A Pilot Study on Healthy Individuals.

BACKGROUND/OBJECTIVES: The development of blood tests for the early detection of individual predisposition to socially significant diseases remains a pressing issue. METHODS: In this pilot study, multiple reaction monitoring mass spectrometry (MRM-MS) with a BAK-270 assay was applied for protein concentrations analysis in blood plasma from 21 healthy volunteers of the European cohort. RESULTS: The levels of 138 plasma proteins were reliably and precisely quantified in no less than 50% of samples. The quantified proteins included 66 FDA-approved markers of cardiovascular diseases (CVD), and other potential biomarkers of pathologies such as cancer, diabetes mellitus, and Alzheimer's disease. The analysis of individual variations of the plasma proteins revealed significant differences between the male (11) and female (10) groups. In total, fifteen proteins had a significantly different concentration in plasma; this included four proteins that exhibited changes greater than ±1.5-fold, three proteins (RBP4, APCS, and TTR) with higher levels in males, and one (SHBG) elevated in females. The obtained results demonstrated considerable agreement with the data collected from 20 samples of a North American cohort, which were analyzed with the similar MRM assay. The most significant differences between the cohorts of the two continents were observed in the level of 42 plasma proteins (including 24 FDA markers), of which 17 proteins showed a ≥1.5-fold change, and included proteins increased in North Americans (APOB, CRTAC1, C1QB, C1QC, C9, CRP, HP, IGHG1, IGKV4-1, SERPING1, RBP4, and AZGP1), as well as those elevated in Europeans (APOF, CD5L, HBG2, SELPLG, and TNA). CONCLUSIONS: The results suggest a different contribution of specific (patho)physiological pathways (e.g., immune system and blood coagulation) to the development of socially significant diseases in Europeans and North Americans, and they should be taken into account when refining diagnostic panels.

Capabilities of MS for analytical quantitative determination of the ratio of α- and ßAsp7 isoforms of the amyloid-ß peptide in binary mixtures.

There is strong evidence that the amyloid-ß peptide (Aß) plays a crucial role in the pathogenesis of Alzheimer's disease (AD), a lethal neurodegenerative disorder of the elderly. During pathology development, the peptide as well as its various chemically modified isoforms is accumulated in specific brain tissues as characteristic proteinaceous deposits, the so-called amyloid plaques, which are the pathomorphological mark of AD, although the level of Αß in the blood is the same for healthy individuals and for AD patients. Earlier, it has been shown that isomerization of aspartate 7, the most abundant post-translational modification of the Αß peptide, is tightly involved in a set of molecular processes associated with AD progression. Therefore, the isoAsp 7-containing Αß isomer (isoAß) is assumed to be a potential biomarker of AD that can be identified in the blood. Here, we present an analytical mass spectrometric method for quantitative determination of the ratio of normal and isomerized Αß fragments 1-16 in their binary mixtures, and all analytical capabilities, such as accuracy, detection limits, and sensitivity of the presented method, are determined and thoroughly discussed. On the basis of this method, an analytical approach for quantitative determination of this modification in the blood will be developed in further studies.

The high-resolution mass spectrometry study of the protein composition of amyloid-like urine aggregates associated with preeclampsia.

The study of protein misfolding and post-translational processing abnormalities is a promising diagnostic approach for socially significant pathologies associated with the accumulation of abnormal forms of proteins. Recently, it was shown that amyloid-like aggregates can be observed in the urine of pregnant women with preeclampsia, which is the most severe hypertensive complication that can lead to fateful outcomes. The protein composition of urine aggregates may clarify the molecular mechanisms underlying the pathology and has not yet been studied in detail. Using a proteomic approach based on high-resolution mass spectrometry, we studied the protein composition of amyloid-like structures that aggregate in the presence of Congo red azo-dye in the urine of pregnant women with preeclampsia. Fragments of ß-sheets of α-1-antitrypsin, complement 3, haptoglobin, ceruloplasmin, and trypstatin were identified as most likely targets for Congo red binding.