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.

A pilot study of anti-nephrin antibodies in podocytopaties among adults.

AIM: Minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS) are podocytopathies characterized by damage to the glomerular filtration barrier, leading to proteinuria and nephrotic syndrome. The production of anti-podocyte antibodies has been proposed as potential circulating factors contributing to the development of these conditions. The aim of the study is to evaluate the levels of anti-nephrin antibodies in patients with podocytopathies and healthy subjects. METHODS: In this study, a total of 77 patients with active glomerulopathy and 11 healthy subjects were included. Forty one patients were diagnosed with FSGS, 11 with MCD, and 25 with MN. To measure the levels of anti-nephrin antibodies, enzyme-linked immunosorbent assay was used. RESULTS: The levels of antibodies to nephrin were significantly higher in patients with MCD 61.2 [28.9-66.3] ng/mL and FSGS 32.5 [17.2-58.4] ng/mL compared to MN 20.3 [14.4-38.4] and healthy individuals 15.3 [12-18.9] ng/mL, p < .05. In patients with primary FSGS, the levels of antibodies to nephrin were significantly higher 45.2 [20-64.3] ng/mL compared to patients with secondary FSGS 26.7 [11.2-44.1] ng/mL, p < .05. There were no significant differences in the remission rate between the anti-nephrin antibodies positive and negative groups (log-rank test: p = .158). CONCLUSION: The level of anti-nephrin antibodies was found to be significantly higher in patients with MCD and pFSGS compared to those with sFSGS, MN, and healthy subjects. Anti-nephrin antibodies in MCD and primary FSGS may be associated with the severity of podocytopathies, however they did not have an impact on the response to therapy.

Quantitative Proteomics of Maternal Blood Plasma in Isolated Intrauterine Growth Restriction.

Intrauterine growth restriction (IUGR) remains a significant concern in modern obstetrics, linked to high neonatal health problems and even death, as well as childhood disability, affecting adult quality of life. The role of maternal and fetus adaptation during adverse pregnancy is still not completely understood. This study aimed to investigate the disturbance in biological processes associated with isolated IUGR via blood plasma proteomics. The levels of 125 maternal plasma proteins were quantified by liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM MS) with corresponding stable isotope-labeled peptide standards (SIS). Thirteen potential markers of IUGR (Gelsolin, Alpha-2-macroglobulin, Apolipoprotein A-IV, Apolipoprotein B-100, Apolipoprotein(a), Adiponectin, Complement C5, Apolipoprotein D, Alpha-1B-glycoprotein, Serum albumin, Fibronectin, Glutathione peroxidase 3, Lipopolysaccharide-binding protein) were found to be inter-connected in a protein-protein network. These proteins are involved in plasma lipoprotein assembly, remodeling, and clearance; lipid metabolism, especially cholesterol and phospholipids; hemostasis, including platelet degranulation; and immune system regulation. Additionally, 18 proteins were specific to a particular type of IUGR (early or late). Distinct patterns in the coagulation and fibrinolysis systems were observed between isolated early- and late-onset IUGR. Our findings highlight the complex interplay of immune and coagulation factors in IUGR and the differences between early- and late-onset IUGR and other placenta-related conditions like PE. Understanding these mechanisms is crucial for developing targeted interventions and improving outcomes for pregnancies affected by IUGR.

Integrating Proteomics and Lipidomics for Evaluating the Risk of Breast Cancer Progression: A Pilot Study.

Metastasis is a serious and often life-threatening condition, representing the leading cause of death among women with breast cancer (BC). Although the current clinical classification of BC is well-established, the addition of minimally invasive laboratory tests based on peripheral blood biomarkers that reflect pathological changes in the body is of utmost importance. In the current study, the serum proteome and lipidome profiles for 50 BC patients with (25) and without (25) metastasis were studied. Targeted proteomic analysis for concertation measurements of 125 proteins in the serum was performed via liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM MS) using the BAK 125 kit (MRM Proteomics Inc., Victoria, BC, Canada). Untargeted label-free lipidomic analysis was performed using liquid chromatography coupled to tandem mass-spectrometry (LC-MS/MS), in both positive and negative ion modes. Finally, 87 serum proteins and 295 lipids were quantified and showed a moderate correlation with tumor grade, histological and biological subtypes, and the number of lymph node metastases. Two highly accurate classifiers that enabled distinguishing between metastatic and non-metastatic BC were developed based on proteomic (accuracy 90%) and lipidomic (accuracy 80%) features. The best classifier (91% sensitivity, 89% specificity, AUC = 0.92) for BC metastasis diagnostics was based on logistic regression and the serum levels of 11 proteins: alpha-2-macroglobulin, coagulation factor XII, adiponectin, leucine-rich alpha-2-glycoprotein, alpha-2-HS-glycoprotein, Ig mu chain C region, apolipoprotein C-IV, carbonic anhydrase 1, apolipoprotein A-II, apolipoprotein C-II and alpha-1-acid glycoprotein 1.

Antitumor Effect of Bleomycin Nanoaerosol in Murine Carcinoma Model.

Bleomycin, which is widely used as an antitumor agent, possesses serious adverse effects such as pulmonary toxicity. Local nanoaerosol deposition for lung cancer treatment is a promising alternative to drug delivery to lung lesions. The aim of this work is to test the hypothesis that bleomycin nanoaerosol can be effectively used to treat multiple lung metastases. To obtain bleomycin nanoaerosol, an aerosol generator based on electrospray of a solution of a nonvolatile substance with gas-phase neutralization of charged aerosol particles was used. Lung metastases in murine Lewis lung carcinoma and B16 melanoma animal models were counted. The effect of inhaled bleomycin nanoparticles on the number and volume of metastases, as well as pulmonary side effects, was investigated. Using a mouse exposure chamber, the dose-dependent effect of inhaled bleomycin on tumor volume was evaluated in comparison with intraperitoneal administration. Bleomycin nanoaerosol reduced the volume of metastases and produced a higher antitumor effect at much lower doses. It has been established that long-term exposure to nanoaerosol with a low dose of bleomycin is capable of suppressing cancer cell growth. The treatment was well tolerated. In the lungs, minor changes were found in the form of focal-diffuse infiltration of the lung parenchyma.

CD44 Expression in Renal Tissue Is Associated with an Increase in Urinary Levels of Complement Components in Chronic Glomerulopathies.

It is suggested that activated CD44+ cells play a profibrogenic role in the pathogenesis of active glomerulopathies. Complement activation is also involved in renal fibrogenesis. The aim of the study was to evaluate the role of the activation of CD44+ cells in the kidney tissue and complement components' filtration to the urine as factors of renal tissue fibrosis in patients with glomerulopathies. In total, 60 patients with active glomerulopathies were included in our study: 29 patients with focal segmental glomerulosclerosis (FSGS), 10 patients with minimal change disease (MCD), 10 patients with membranous nephropathy (MN), and 11 patients with IgA nephropathy. The immunohistochemical peroxidase method was used to study the expression of CD44+ in kidney biopsies. Components of complement were analyzed in urine by the multiple reaction monitoring (MRM) approach using liquid chromatography. Strong CD44 expression was noted predominantly in PEC and mesangial cells (MC) in patients with FSGS, and to a lesser extent, in patients with MN and IgA nephropathy, and it was absent in patients with MCD. Expression of profibrogenic CD44+ in glomeruli correlated with the levels of proteinuria and complement C2, C3, and C9 components, and CFB and CFI in urine. The CD44+ expression scores in the renal interstitium correlated with the level of C3 and C9 components of complement in the urine and the area of tubulo-interstitial fibrosis. The strongest expression of CD44+ was found in the glomeruli (MC, PEC, and podocytes) of patients with FSGS compared with other glomerulopathies. The CD44 expression score in the glomeruli and interstitium is associated with high levels of complement components in the urine and renal fibrosis.

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).

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.

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.

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.

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.

Combined Impact of Magnetic Force and Spaceflight Conditions on Escherichia coli Physiology.

Changes in bacterial physiology caused by the combined action of the magnetic force and microgravity were studied in Escherichia coli grown using a specially developed device aboard the International Space Station. The morphology and metabolism of E. coli grown under spaceflight (SF) or combined spaceflight and magnetic force (SF + MF) conditions were compared with ground cultivated bacteria grown under standard (control) or magnetic force (MF) conditions. SF, SF + MF, and MF conditions provided the up-regulation of Ag43 auto-transporter and cell auto-aggregation. The magnetic force caused visible clustering of non-sedimenting bacteria that formed matrix-containing aggregates under SF + MF and MF conditions. Cell auto-aggregation was accompanied by up-regulation of glyoxylate shunt enzymes and Vitamin B12 transporter BtuB. Under SF and SF + MF but not MF conditions nutrition and oxygen limitations were manifested by the down-regulation of glycolysis and TCA enzymes and the up-regulation of methylglyoxal bypass. Bacteria grown under combined SF + MF conditions demonstrated superior up-regulation of enzymes of the methylglyoxal bypass and down-regulation of glycolysis and TCA enzymes compared to SF conditions, suggesting that the magnetic force strengthened the effects of microgravity on the bacterial metabolism. This strengthening appeared to be due to magnetic force-dependent bacterial clustering within a small volume that reinforced the effects of the microgravity-driven absence of convectional flows.

Blood Plasma Proteins Associated With Heart Rate Variability in Cosmonauts Who Have Completed Long-Duration Space Missions.

The study presents the results of evaluating the changes in the concentrations of blood plasma proteins associated with heart rate variability (HRV) in cosmonauts who have completed space missions lasting about 6months. The concentrations of 125 proteins were quantified in biological samples of the cosmonauts' blood plasma. The subgroups of proteins associated with the physiological processes of the HRV autonomic regulation were identified using bioinformatic resources (Immunoglobulin heavy constant mu, Complement C1q subcomponent subunit C, Plasma serine protease inhibitor, Protein-72kDa type IV collagenase, Fibulin-1, Immunoglobulin lambda constant 3). The concentration of these proteins in the blood plasma before the flight, and the dynamics of concentration changes on the 1st and 7th days of the post-flight rehabilitation period differed in the groups of cosmonauts with a predominance of sympathetic or parasympathetic modulating autonomous influences. The dynamics of changes in the concentrations of the identified set of proteins reveal that in cosmonauts with a predominance of sympathetic modulating influences, the mechanisms of autonomic regulation are exposed to significant stress in the recovery period immediately after the completion of the space mission, compared with the cosmonauts with a predominance of parasympathetic modulating influences.

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.

Mass spectrometry based proteome profiling of the exhaled breath condensate for lung cancer biomarkers search.

INTRODUCTION: Lung cancer remains the most prevalent cause of cancer mortality worldwide mainly due to insufficient availability of early screening methods for wide-scale application. Exhaled breath condensate (EBC) is currently considered as one of the promising targets for early screening and is particularly attractive due to its absolutely noninvasive collection and possibility for long-term frozen storage. EBC proteome analysis can provide valuable information about the (patho)physiological changes in the respiratory system and may help to identify in time a high risk of lung cancer. Mass spectrometry (MS) profiling of EBC proteome seems to have no alternative in obtaining the most extensive data and characteristic marker panels for screening. AREAS COVERED: This special report summarizes the data of several proteomic studies of EBC in normal and lung cancer (from 2012 to 2021, PubMed), focuses on the possible reasons for the significant discrepancy in the results, and discusses some aspects for special attention in further studies. EXPERT OPINION: The significant discrepancy in the results of various studies primarily highlights the need to create standardized protocols for the collection and preparation of EBC for proteomic analysis. The application of quantitative and targeted LC-MS/MS based approaches seems to be the most promising in further EBC proteomic studies.

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.

Normalization methods for reducing interbatch effect without quality control samples in liquid chromatography-mass spectrometry-based studies.

Data normalization is an essential part of a large-scale untargeted mass spectrometry metabolomics analysis. Autoscaling, Pareto scaling, range scaling, and level scaling methods for liquid chromatography-mass spectrometry data processing were compared with the most common normalization methods, including quantile normalization, probabilistic quotient normalization, and variance stabilizing normalization. These methods were tested on eight datasets from various clinical studies. The efficiency of the data normalization was assessed by the distance between clusters corresponding to batches and the distance between clusters corresponding to clinical groups in the space of principal components, as well as by the number of features with a pairwise statistically significant difference between the batches and the number of features with a pairwise statistically significant difference between clinical groups. Autoscaling demonstrated the most effective reduction in interbatch variation and can be preferable to probabilistic quotient or quantile normalization in liquid chromatography-mass spectrometry data.

Comparison of the effectiveness of variable selection method for creating a diagnostic panel of biomarkers for mass spectrometric lipidome analysis.

Hundreds of compounds are detected during untargeted lipidomics analysis. The potential efficacy of lipids as disease markers makes it important to select the species with the most discriminative potential. Datasets based on a selected class of lipids allow the development of a high-quality diagnostic model using orthogonal projection on latent structure. The combination of selection of lipids by variable importance in projection and by Akaike information criteria makes it possible to build a reliable diagnostic model based on logistic regression.

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.