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Owing to their close proximity, pericardial fluid (PF)'s proteome may mirror the pathophysiological status of the heart. Despite this diagnosis potential, the knowledge of PF's proteome is scarce. Large amounts of albumin hamper the characterization of the least abundant proteins in PF. Aiming to expand PF's proteome and to validate the technique for future applications, we have fractionated and characterized the PF, using N-(trimethoxysilylpropyl)ethylenediamine triacetic acid (EDTA)-functionalized magnetic nanoparticles (NPs@EDTA) followed by a GeLC-MS/MS approach. Similarly to an albumin-depletion kit, NPs@EDTA-based fractionation was efficient in removing albumin. Both methods displayed comparable inter-individual variability, but NPs@EDTA outperformed the former with regard to the protein dynamic range as well as to the monitoring of biological processes. Overall, 565 proteins were identified, of which 297 (>50%) have never been assigned to PF. Moreover, owing to this method's good proteome reproducibility, affordability, rapid automation and high binding ability of NP@EDTA, it bears a great potential towards future clinical application., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The efficiency of proteostasis regulation declines during aging and the failure of protein homeostasis is common in age-related diseases. Protein oxidation is a major contributor to the loss of proteome homeostasis, also called "proteostasis," precluding protein misfolding and aggregation. So, the identification of the molecular pathways impaired by protein oxidation will increase the understanding of proteostasis and the pathophysiological conditions related to the loss of proteostasis. Sample derivatization with dinitrophenyl hydrazine and western blot immunoassay detection of carbonylated proteins (commonly known as Oxyblot™) coupled to mass spectrometry (blot-MS) is an attractive methodological approach to identify proteins that are more prone to carbonylation, a typical oxidative modification of amino acid residues. The integration of blot-MS data of carbonylated proteins with bioinformatics tools allows the identification of the biological processes more affected by protein oxidation and that, eventually, result in the loss of proteostasis.In this chapter, we describe a blot-MS methodology to identify the proteins more prone to oxidation in biological samples, as cell and tissue extracts, and biofluids. Analysis of mitochondria isolated from cardiac tissue is provided as an example. Bioinformatic strategy to deal with data retrieved from blot-MS experiments are proposed for the identification of relevant biological processes modulated by oxidative stress stimuli.

The worldwide number of individuals suffering from diabetes mellitus (DM) has been projected to rise from 171 million in 2000 to 366 million in 2030. Identification of specific biomarkers for prediction and monitoring of DM is needed not only for the adequate screening diagnosis but also to assist the design of interventions to prevent or delay progression of this pathology and its attendant complications. Proteomic methods based on MS hold special promise for the identification of novel biomarkers that might form the foundation for new clinical tests, but to date, their contribution has been somehow unfruitful. Indeed, from more than 300 proteins found differently modulated in body fluids from diabetic patients, approximately 50 were validated with other approaches like ELISA or Western blotting and the clinical trials are being initiated to employ biofluids' proteomics (specifically urinary proteomics) in clinical decision. This review provides an overview of MS-based applications in the identification of potential biomarkers for DM, emphasizing the methodological challenges involved., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Saliva has gained increasing attention in the quest for disease biomarkers. Because it is a biological fluid that can be collected is an easy, painless, and safe way, it has been increasingly studied for the identification of oral cancer biomarkers. This is particularly important because oral cancer is often diagnosed at late stages with a poor prognosis. The review addresses the evolution of the experimental approaches used in salivary proteomics studies of oral cancer over the years and outlines advantages and pitfalls related to each one. In addition, examines the current landscape of oral cancer biomarker discovery and translation focusing on salivary proteomic studies. This discussion is based on an extensive literature search (PubMed, Scopus and Google Scholar). The introduction of mass spectrometry has revolutionized the study of salivary proteomics. In the future, the focus will be on refining existing methods and introducing powerful experimental techniques such as mass spectrometry with selected reaction monitoring, which, despite their effectiveness, are still underutilized due to their high cost. In addition, conducting studies with larger cohorts and establishing standardized protocols for salivary proteomics are key challenges that need to be addressed in the coming years. [ABSTRACT FROM AUTHOR]

Bladder cancer (BCa) research relying on Omics approaches has increased over the last few decades, improving the understanding of BCa pathology and contributing to a better molecular classification of BCa subtypes. To gain further insight into the molecular profile underlying the development of BCa, a systematic literature search was performed in PubMed until November 2023, following the PRISMA guidelines. This search enabled the identification of 25 experimental studies using mass spectrometry or nuclear magnetic resonance-based approaches to characterize the metabolite signature associated with BCa. A total of 1562 metabolites were identified to be altered by BCa in different types of samples. Urine samples displayed a higher likelihood of containing metabolites that are also present in bladder tumor tissue and cell line cultures. The data from these comparisons suggest that increased concentrations of L-isoleucine, L-carnitine, oleamide, palmitamide, arachidonic acid and glycoursodeoxycholic acid and decreased content of deoxycytidine, 5-aminolevulinic acid and pantothenic acid should be considered components of a BCa metabolome signature. Overall, molecular profiling of biological samples by metabolomics is a promising approach to identifying potential biomarkers for early diagnosis of different BCa subtypes. However, future studies are needed to understand its biological significance in the context of BCa and to validate its clinical application. [ABSTRACT FROM AUTHOR]

Simple Summary: Prostate cancer (PCa) is one of the most common cancers. Due to the limited and invasive approaches for PCa diagnosis, it is crucial to identify more accurate and non-invasive biomarkers for its detection. The aim of our study was to non-invasively uncover new protein targets for detecting PCa using a proteomics and proteogenomics approach. This work identified several dysregulated mutant protein isoforms in urine from PCa patients, some of them predicted to have a protective or an adverse role in these patients. These results are promising given urine's non-invasive nature and offers an auspicious opportunity for research and development of PCa biomarkers. To identify new protein targets for PCa detection, first, a shotgun discovery experiment was performed to characterize the urinary proteome of PCa patients. This revealed 18 differentially abundant urinary proteins in PCa patients. Second, selected targets were clinically tested by immunoblot, and the soluble E-cadherin fragment was detected for the first time in the urine of PCa patients. Third, the proteogenome landscape of these PCa patients was characterized, revealing 1665 mutant protein isoforms. Statistical analysis revealed 6 differentially abundant mutant protein isoforms in PCa patients. Analysis of the likely effects of mutations on protein function and PPIs involving the dysregulated mutant protein isoforms suggests a protective role of mutations HSPG2*Q1062H and VASN*R161Q and an adverse role of AMBP*A286G and CD55*S162L in PCa patients. This work originally characterized the urinary proteome, focusing on the proteogenome profile of PCa patients, which is usually overlooked in the analysis of PCa and body fluids. Combined analysis of mass spectrometry data using two different software packages was performed for the first time in the context of PCa, which increased the robustness of the data analysis. The application of proteogenomics to urine proteomic analysis can be very enriching in mutation-related diseases such as cancer. [ABSTRACT FROM AUTHOR]

• Targeted screening approach (PIS 97) was applied to monitor sulfate-based lipids in human biofluids and cells. • Sulfate-based lipids (SL) in lipid extracts included steroid sulfates, sulfoglycolipids and other SL. • Other SL included N-acyl taurine, hypurate conjugates, and polyphenol metabolites. • Lipid extracts exhibit a heterogeneous distribution of SL across human fluids and cells. • Sulphur/phosphorous estimates show seminal fluid and epithelial cells as highly enriched in SL. Sulfate-based lipids (SL) have been proposed as players in inflammation, immunity and infection. In spite of the many biochemical processes linked to SL, analysis on this class of lipids has only focused on specific SL sub-classes in individual fluids or cells leaving a range of additional SL in other biological samples unaccounted for. This study describes the mass spectrometry screening of SL in lipid extracts of human fluids (saliva, plasma, urine, seminal fluid) and primary human cells (RBC, neutrophils, fibroblasts and skin epidermal) using targeted precursor ion scanning (PIS) approach. The PIS 97 mass spectra reveal a wide diversity of SL including steroid sulfates, sulfoglycolipids and other unidentified SL, as well as metabolites such as taurines, sulfated polyphenols and hypurate conjugates. Semi-quantification of SL revealed that plasma exhibited the highest content of SL whereas seminal fluid and epithelial cells contained the highest sulphur to phosphorous (S/P) ratio. The complexity of biofluids and cells sulfateome presented in this study highlight the importance of expanding the panel of synthetic sulfate-based lipid standards. Also, the heterogenous distribution of SL provides evidence for the interplay of sulfotransferases/sulfatases, opening new avenues for biomarker discovery in oral health, cardiovascular, fertility and dermatology research areas. [ABSTRACT FROM AUTHOR]

Cerebrospinal fluid (CSF) is an excellent source of biological information regarding the nervous system, once it is in close contact and accurately reflects alterations in this system. Several studies have analyzed differential protein profiles of CSF samples between healthy and diseased human subjects. However, the pathophysiological mechanisms and how CSF proteins relate to diseases are still poorly known. By applying bioinformatics tools, we attempted to provide new insights on the biological and functional meaning of proteomics data envisioning the identification of putative disease biomarkers. Bioinformatics analysis of data retrieved from 99 mass spectrometry (MS)-based studies on CSF profiling highlighted 1985 differentially expressed proteins across 49 diseases. A large percentage of the modulated proteins originate from exosome vesicles, and the majority are involved in either neuronal cell growth, development, maturation, migration, or neurotransmitter-mediated cellular communication. Nevertheless, some diseases present a unique CSF proteome profile, which were critically analyzed in the present study. For instance, 48 proteins were found exclusively upregulated in the CSF of patients with Alzheimer’s disease and are mainly involved in steroid esterification and protein activation cascade processes. A higher number of exclusively upregulated proteins were found in the CSF of patients with multiple sclerosis (76 proteins) and with bacterial meningitis (70 proteins). Whereas in multiple sclerosis, these proteins are mostly involved in the regulation of RNA metabolism and apoptosis, in bacterial meningitis the exclusively upregulated proteins participate in inflammation and antibacterial humoral response, reflecting disease pathogenesis. The exploration of the contribution of exclusively upregulated proteins to disease pathogenesis will certainly help to envision potential biomarkers in the CSF for the clinical management of nervous system diseases. [ABSTRACT FROM PUBLISHER]

Growing evidence suggests that a range of reversible protein post-translational modifications such as acetylation regulates mitochondria signalling, impacting cellular homeostasis. However, the extent of this type of regulation in the control of mitochondria functionality is just beginning to be discovered, aided by the availability of high-resolution mass spectrometers and bioinformatic tools. Data mining from literature on protein acetylation profiling focused on mitochondria isolated from tissues retrieved more than 1395 distinct proteins, corresponding to more than 4858 acetylation sites. ClueGo analysis of identified proteins highlighted oxidative phosphorylation, tricarboxylic acid cycle, fatty acid oxidation and amino acid metabolism as the biological processes more prone to regulation through acetylation. This review also examines the physiological relevance of protein acetylation on the molecular pathways harbored in mitochondria under distinct pathophysiological conditions as caloric restriction and alcohol-induced liver damage. This integrative perspective will certainly help to envisage future studies targeting the regulation of mitochondrial functionality. [ABSTRACT FROM AUTHOR]

ABSTRACT Diabetic patients show a high susceptibility to oral diseases of inflammatory, catabolic and chronic nature with potential impact on saliva composition. In this study, our purpose was to characterize type 1 diabetes-induced alterations in the salivary peptidome aiming to find prospective biomarkers for type 1 diabetes oral health evaluation. Peptidomic analysis of saliva from controls ( n = 5) and type 1 diabetic patients ( n = 5) were performed by liquid chromatography followed by mass spectrometry. The proteolytic activity and metalloproteinases expression was accessed by zymography and slot blot analysis, respectively. Data evidenced a significant increase in the percentage of peptides in diabetic patients paralleled by a higher proteolytic activity, compared with healthy individuals. The nonsalivary gland protein fragments identified in saliva were mainly derived from collagen and extracellular matrix proteins, namely collagen type I. The cleavage site frequency analysis showed significant differences between healthy and type 1 diabetic individuals, highlighting the activity of proteases such as matrix metalloproteinase-9 and cathepsin D. Our results highlight salivary collagen fragments as potential biomarkers to follow up diabetes-related oral damage. Copyright © 2011 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

In the present study, a proteomic approach was applied to evaluate the influence of salivary protein composition on in vitro dental pellicle formation and its possible correlation with dental caries. Whole saliva, collected from caries-free and caries-susceptible subjects, was analyzed by two-dimensional electrophoresis, and protein spots were identified by mass spectrometry. Data analysis of salivary protein composition showed a statistically significant correlation between the quantity of acidic proline-rich proteins (PRPs) , lipocalin, cystatin SN and cystatin S, and samples from the caries-free group of subjects [decayed, missing or filled teeth (DMFT) = 0]. Samples from subjects with a high DMFT index appear to be correlated with high levels of amylase, immunoglobulin A, and lactoferrin. In vitro pellicle-composition experiments showed the same correlations found for whole saliva. As cystatins are known physiological inhibitors of cathepsins, the higher quantities of lipocalin, and cystatins S and SN found in the samples from the caries-free subjects suggest that inhibition of proteolytic events on other salivary proteins may indirectly provide tooth protection. The correlation between higher levels of the phosphorylated acidic PRPs 1/2 with samples from the caries-free group also suggests a protective role for these proteins. [ABSTRACT FROM AUTHOR]

Urine is a highly attractive source of biological information and disease biomarkers, whose proteome characterization is ongoing. To that end, depletion/enrichment strategies for protein analysis can be of great convenience. We have thus developed a method based on the use of EDTA-functionalized magnetic nanoparticles (NPs@EDTA), to fractionate urine samples before liquid chromatography-mass spectrometry analysis and compared the identified proteins with those obtained from ultrafiltrated/unfractionated (UF) urine samples. NPs@EDTA allowed larger urine volumes to be processed, resulting in a greater number of protein identifications (~2-fold) at a lower cost when compared to UF samples. Proteins of greater abundance (such as albumin and uromodulin) were, at least partially, depleted with NPs@EDTA while those of lower abundance were enriched. Bioinformatics analysis showed that approximately 27% of NPs@EDTA-enriched proteins were annotated as displaying enzymatic activity, most of these being hydrolytic enzymes (56%), particularly proteases/peptidases (48%). Also, post-translational modifications were prominently predicted across NPs@EDTA-enriched proteins (90%), particularly glycosylation (52%), phosphorylation (47%) and acetylation (30%). NPs@EDTA allowed the identification of 109 proteins in urine for the first time, showing high potential as a platform for urine's fractionation prior to proteomic analysis. [ABSTRACT FROM AUTHOR]

A new chelating chromatography method was developed based in the use of magnetic iron oxide nanoparticles functionalized with EDTA-TMS ((N-(trimethoxysilylpropyl)ethylenediaminetriacetate trisodium salt). These particles combine a high surface area, biocompatibility and magnetic removal from solution, with the chelating affinity towards metal ions. The particles were used to selectively capture metallo-dependant proteins in secretome obtained from human monocytes and mouse macrophages. Secreted metallo-dependant proteins are highly important sources of information since they are involved in several pathological processes. The identification of secreted proteins involved in these processes is highly important for diagnosis or monitoring the progression of a disease. In this multiple-approach study it was possible to not only selectively capture several secreted metallo-dependant proteins, but also to significantly avoid masking proteins such as the highly abundant albumin form the fetal bovine serum used to supplement the cell culture medium. Overall, the magnetic nanoparticle-based chelating chromatography method developed here has proved to be a sensitive, low cost, and a quick tool for sample treatment in order to selectively enrich metalloproteins while overcoming the contamination of highly abundant proteins. Graphical abstractfx1Highlights· Development of a new chelating chromatography method. · A quick tool for sample treatment to selectively enrich metalloproteins.. · Highly abundant proteins such as albumin are not captured by this system. [ABSTRACT FROM AUTHOR]

In recent years, there has been an increasing interest in nitrofatty acids (NO2-FA) as signaling molecules formed under nitroxidative stress. NO2-FA were detected in vivo in a free form, although it is assumed that they may also be esterified to phospholipids (PL). Nevertheless, insufficient discussion about the nature, origin, or role of nitro phospholipids (NO2-PL) was reported up to now. The aim of this study was to develop a mass spectrometry (MS) based approach which allows identifying nitroalkenes derivatives of three major PL classes found in living systems: phosphatidylcholines (PCs), phosphatidylethanolamine (PEs), and phosphatidylserines (PSs). NO2-PLs were generated by NO2BF4 in hydrophobic environment, mimicking biological systems. The NO2-PLs were then detected by electrospray ionization (ESI-MS) and ESI-MS coupled to hydrophilic interaction liquid chromatography (HILIC). Identified NO2-PLs were further analyzed by tandem MS in positive (as [M + H]+ ions for all PL classes) and negative-ion mode (as [M - H]- ions for PEs and PSs and [M + OAc]- ions for PCs). Typical MS/MS fragmentation pattern of all NO2-PL included a neutral loss of HNO2, product ions arising from the combined loss of polar headgroup and HNO2, [NO2-FA + H]+ and [NO2-FA - H]- product ions, and cleavages on the fatty acid backbone near the nitro group, allowing its localization within the FA akyl chain. Developed MS method was used to identify NO2-PL in cardiac mitochondria from a well-characterized animal model of type 1 diabetes mellitus. We identified nine NO2-PCs and one NO2-PE species. The physiological relevance of these findings is still unknown. [ABSTRACT FROM AUTHOR]

Highlights: [•] T1DM is associated with changes in liver lipid profile. [•] Relative contents of PC, LPC and SM decrease, while the other PL classes increase. [•] Remodeling of acyl chains comprises reduction of 16:0 and increase of 18:0 and 20:4. [•] PL changes seem to underlie liver dysfunction related with type 1 diabetes mellitus. [ABSTRACT FROM AUTHOR]

Despite the large number of studies focused on the impact of diseases on urine proteome, few outputs with clinical meaning were retrieved so far. The goal of this study was to identify the biological processes modulated in urine by distinct diseases to better understand disease pathogenesis and to identify urinary proteins with potential diagnosis value. We searched PubMed and SCOPUS databases for mass spectrometry-based experimental papers and pooled differentially expressed proteins by disease and target organic system. A total of 2572 differentially expressed proteins or peptides were pooled from 89 studies focused on 57 diseases. Data analysis highlighted inflammation as a biological process modulated by all diseases. However, specific inflammatory signatures are associated with specific diseases and/or aetiologies. Moreover, specific biological processes were identified for specific groups of pathologies and unique proteins identified. Overall, integrative data analysis from urine proteomics/peptidomics reinforces the clinical potential of this body fluid for the clinical management of distinct diseases. [ABSTRACT FROM AUTHOR]

Infectious diseases caused by multidrug-resistant (MDR) Enterobacteriaceae have exponentially increased in the past decade, and are a major concern in hospitals. In the first part of the work, we compared the proteome profile of MDR and susceptible clinical isolates of Escherichia coli and Klebsiella pneumoniae in order to identify possible biological processes associated with drug resistance and susceptible phenotypes, using a label-free approach. In the second part, we used an immunoproteomics approach to identify immunoreactive proteins in the same isolates. A total of 388 and 377 proteins were identified in MDR and susceptible E. coli , respectively, evidencing that biological processes related to translation are upregulated in E. coli MDR, while there is an upregulation of processes related to catalytic activity in K. pneumoniae MDR. Both MDR strains show downregulation of processes related to amino acid activation and tRNA amino-acylation. Our data also suggest that MDR strains have higher immunoreactivity than the susceptible strains. The application of high-throughput mass spectrometry (MS) and bioinformatics to the study of modulation of biological processes might shed light on the characterization of multidrug resistance in bacteria. [ABSTRACT FROM AUTHOR]