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Uterine fibroids (UF) is the most common (about 70% cases) type of gynecological disease, with the recurrence rate varying from 11 to 40%. Because UF has no distinct symptomatology and is often asymptomatic, the specific and sensitive diagnosis of UF as well as the assessment for the probability of UF recurrence pose considerable challenge. The aim of this study was to characterize alterations in the lipid profile of tissues associated with the first-time diagnosed UF and recurrent uterine fibroids (RUF) and to explore the potential of mass spectrometry (MS) lipidomics analysis of blood plasma samples for the sensitive and specific determination of UF and RUF with low invasiveness of analysis. MS analysis of lipid levels in the myometrium tissues, fibroids tissues and blood plasma samples was carried out on 66 patients, including 35 patients with first-time diagnosed UF and 31 patients with RUF. The control group consisted of 15 patients who underwent surgical treatment for the intrauterine septum. Fibroids and myometrium tissue samples were analyzed using direct MS approach. Blood plasma samples were analyzed using high performance liquid chromatography hyphened with mass spectrometry (HPLC/MS). MS data were processed by discriminant analysis with projection into latent structures (OPLS-DA). Significant differences were found between the first-time UF, RUF and control group in the levels of lipids involved in the metabolism of glycerophospholipids, sphingolipids, lipids with an ether bond, triglycerides and fatty acids. Significant differences between the control group and the groups with UF and RUF were found in the blood plasma levels of cholesterol esters, triacylglycerols, (lyso) phosphatidylcholines and sphingomyelins. Significant differences between the UF and RUF groups were found in the blood plasma levels of cholesterol esters, phosphotidylcholines, sphingomyelins and triacylglycerols. Diagnostic models based on the selected differential lipids using logistic regression showed sensitivity and specificity of 88% and 86% for the diagnosis of first-time UF and 95% and 79% for RUF, accordingly. This study confirms the involvement of lipids in the pathogenesis of uterine fibroids. A diagnostically significant panel of differential lipid species has been identified for the diagnosis of UF and RUF by low-invasive blood plasma analysis. The developed diagnostic models demonstrated high potential for clinical use and further research in this direction.

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.

Current methods for the intraoperative determination of breast cancer margins commonly suffer from the insufficient accuracy, specificity and/or low speed of analysis, increasing the time and cost of operation as well the risk of cancer recurrence. The purpose of this study is to develop a method for the rapid and accurate determination of breast cancer margins using direct molecular profiling by mass spectrometry (MS). Direct molecular fingerprinting of tiny pieces of breast tissue (approximately 1 &times, 1 &times, 1 mm) is performed using a home-built tissue spray ionization source installed on a Maxis Impact quadrupole time-of-flight mass spectrometer (qTOF MS) (Bruker Daltonics, Hamburg, Germany). Statistical analysis of MS data from 50 samples of both normal and cancer tissue (from 25 patients) was performed using orthogonal projections onto latent structures discriminant analysis (OPLS-DA). Additionally, the results of OPLS classification of new 19 pieces of two tissue samples were compared with the results of histological analysis performed on the same tissues samples. The average time of analysis for one sample was about 5 min. Positive and negative ionization modes are used to provide complementary information and to find out the most informative method for a breast tissue classification. The analysis provides information on 11 lipid classes. OPLS-DA models are created for the classification of normal and cancer tissue based on the various datasets: All mass spectrometric peaks over 300 counts, peaks with a statistically significant difference of intensity determined by the Mann&ndash, Whitney U-test (p &lt, 0.05), peaks identified as lipids, both identified and significantly different peaks. The highest values of Q2 have models built on all MS peaks and on significantly different peaks. While such models are useful for classification itself, they are of less value for building explanatory mechanisms of pathophysiology and providing a pathway analysis. Models based on identified peaks are preferable from this point of view. Results obtained by OPLS-DA classification of the tissue spray MS data of a new sample set (n = 19) revealed 100% sensitivity and specificity when compared to histological analysis, the &ldquo, gold&rdquo, standard for tissue classification. &ldquo, All peaks&rdquo, and &ldquo, significantly different peaks&rdquo, datasets in the positive ion mode were ideal for breast cancer tissue classification. Our results indicate the potential of tissue spray mass spectrometry for rapid, accurate and intraoperative diagnostics of breast cancer tissue as a means to reduce surgical intervention.

Detection of viral RNA by PCR is currently the main diagnostic tool for COVID-19 [1]. The PCR-based test, however, shows limited sensitivity, especially at early and late stages of the disease development [2,3], and is relatively time consuming. Fast and reliable complementary methods for detecting the viral infection would be of help in the current pandemia 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. The N protein of the SARS-COV-2 virus, the most abundant protein in the virion, is the best candidate for mass-spectrometric detection of the infection, and MS-based detection of several peptides from the SARS-COoV-2 nucleoprotein has been reported earlier by the Sinz group [4]. Our approach shows confident identification of the N protein in patient samples even with the lowest viral loads and a much simpler preparation procedure. Our main protocol consists of virus inactivation by heating and adding 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.

Real-time molecular navigation of tissue surgeries is an important goal at present. Combination of electrosurgical units and mass spectrometry (MS) to perform accurate molecular visualization of biological tissues has been pursued by many research groups. Determination of molecular tissue composition at a particular location by surgical smoke analysis is now of increasing interest for clinical use. However, molecular analysis of surgical smoke is commonly lacking molecular specificity and is associated with significant carbonization and chemical contamination, which are mainly related to the high temperature of smoke at which many molecules become unstable. Unlike traditional electrosurgical tools, low-temperature electrosurgical units allow tissue dissection without substantial heating. Here, we show that low-temperature electrosurgical units can be used for desorption of molecules from biological tissues without thermal degradation. The use of extractive electrospray ionization technique for the ionization of desorbed molecules allowed us to obtain mass spectra of healthy and pathological tissues with high degree of differentiation. Overall, the data indicate that the described approach has potential for intraoperative use.

The mass spectrometry-based molecular profiling can be used for better differentiation between normal and cancer tissues and for the detection of neoplastic transformation, which is of great importance for diagnostics of a pathology, prognosis of its evolution trend, and development of a treatment strategy. The aim of the present study is the evaluation of tissue classification approaches based on various data sets derived from the molecular profile of the organic solvent extracts of a tissue. A set of possibilities are considered for the orthogonal projections to latent structures discriminant analysis: all mass spectrometric peaks over 300 counts threshold, subset of peaks selected by ranking with support vector machine algorithm, peaks selected by random forest algorithm, peaks with the statistically significant difference of the intensity determined by the Mann-Whitney U test, peaks identified as lipids, and both identified and significantly different peaks. The best predictive potential is obtained for OPLS-DA model built on nonpolar glycerolipids (Q2 = 0.64, area under curve [AUC] = 0.95); the second one is OPLS-DA model with lipid peaks selected by random forest algorithm (Q2 = 0.58, AUC = 0.87). Moreover, models based on particular molecular classes are more preferable from biological point of view, resulting in new explanatory mechanisms of pathophysiology and providing a pathway analysis. Another promising features for OPLS-DA modeling are phosphatidylethanolamines (Q2 = 0.48, AUC = 0.86).

Cervicovaginal fluid (CVF) is a valuable source of clinical information about the female reproductive tract in both nonpregnant and pregnant women. The aim of this study is to specify the CVF proteome at different stages of cervix neoplastic transformation by label-free quantitation approach based on liquid chromatography tandem mass spectrometry (LC-MS/MS) method. The proteome composition of CVF from 40 women of reproductive age with human papillomavirus (HPV)-associated cervix neoplastic transformation (low-grade squamous intraepithelial lesion [LSIL], high-grade squamous intraepithelial lesion [HSIL], and CANCER) was investigated. Hierarchical clustering and principal component analysis (PCA) of the proteomic data obtained by a label-free quantitation approach show the distribution of the sample set between four major clusters (no intraepithelial lesion or malignancy [NILM], LSIL, HSIL and CANCER) depending on the form of cervical lesion. Multisample ANOVA with subsequent Welch's t test resulted in 117 that changed significantly across the four clinical stages, including 27 proteins significantly changed in cervical cancer. Some of them were indicated as promising biomarkers previously (ACTN4, VTN, ANXA1, CAP1, ANXA2, and MUC5B). CVF proteomic data from the discovery stage were analyzed by the partial least squares-discriminant analysis (PLS-DA) method to build a statistical model, allowing to differentiate severe dysplasia (HSIL and CANCER) from the mild/normal stage (NILM and LSIL), and receiver operating characteristic (ROC) area under the curve (AUC) were obtained on an independent set of 33 samples. The sensitivity of the model was 77%, and the specificity was 94%; AUC was equal to 0.87. CVF proteome proved to be reflect the stage of cervical epithelium neoplastic process.

Specifics of urine proteome is sensitive to a multitude of factors. One is nutrition or entrance in organism of main nutrients including salt (NaCI). Purpose of the investigation was to study the proteomic composition of healthy human urine in the controlled environment of a 105-day isolation experiment (projectMars-500) with various levels of salt intake. Analysis was performed using the present-day proteomics techniques based on chromatography-mass spectrometry and bio-informatics options. An attempt was made to correlate changes in processes and physiological systems with the controlled salt intake. As a result, a list of proteins directly responsible for different salt intake during the experiment and then a list of tissues where these proteins express predominantly were compiled; besides, analysis of the processes these proteins are involved in was performed.

Urine is a sample of choice for noninvasive biomarkers search because it is easily available in large amounts and its molecular composition provides information on processes in the organism. The high potential of urine peptidomics has been demonstrated for clinical purpose. Several mass spectrometry based approaches have been successfully applied for urine peptidome analysis and potential biomarkers search. Summarizing literature data and our own experience we developed a protocol for comprehensive urine peptidome analysis. The technology includes several stages and consists of urine sample preparation by size exclusion chromatography and identification of featured peptides by nano-HPLC coupled to Fourier transform ion cyclotron resonance mass spectrometry, semiquantitative and statistical data analysis.

Recent research revealed that tissue spray mass spectrometry enables rapid molecular profiling of biological tissues, which is of great importance for the search of disease biomarkers as well as for online surgery control. However, the payback for the high speed of analysis in tissue spray analysis is the generally lower chemical sensitivity compared with the traditional approach based on the offline chemical extraction and electrospray ionization mass spectrometry detection. In this study, high resolution mass spectrometry analysis of endometrium tissues of different localizations obtained using direct tissue spray mass spectrometry in positive ion mode is compared with the results of electrospray ionization analysis of lipid extracts. Identified features in both cases belong to three lipid classes: phosphatidylcholines, phosphoethanolamines, and sphingomyelins. Lipids coverage is validated by hydrophilic interaction liquid chromatography with mass spectrometry of lipid extracts. Multivariate analysis of data from both methods reveals satisfactory differentiation of eutopic and ectopic endometrium tissues. Overall, our results indicate that the chemical information provided by tissue spray ionization is sufficient to allow differentiation of endometrial tissues by localization with similar reliability but higher speed than in the traditional approach relying on offline extraction. Graphical Abstract ᅟ.

Mesenchymal stem cells (MSCs) have emerged as a potent therapeutic tool for the treatment of a number of pathologies, including immune pathologies. However, unwelcome effects of MSCs on blood coagulation have been reported, motivating us to explore the thrombotic properties of human MSCs from the umbilical cord. We revealed strong procoagulant effects of MSCs on human blood and platelet-free plasma using rotational thromboelastometry and thrombodynamic tests. A similar potentiation of clotting was demonstrated for MSC-derived extracellular vesicles (EVs). To offer approaches to avoid unwanted effects, we studied the impact of a heparin supplement on MSC procoagulative properties. However, MSCs still retained procoagulant activity toward blood from children receiving a therapeutic dose of unfractionated heparin. An analysis of the mechanisms responsible for the procoagulant effect of MSCs/EVs revealed the presence of tissue factor and other proteins involved in coagulation-associated pathways. Also, we found that some MSCs and EVs were positive for annexin V, which implies the presence of phosphatidylserine on their surfaces, which can potentiate clot formation. Thus, we revealed procoagulant activity of MSCs/EVs associated with the presence of phosphatidylserine and tissue factor, which requires further analysis to avoid adverse effects of MSC therapy in patients with a risk of thrombosis.

Obtaining fast screening information on molecular composition of a tissue sample is of great importance for a disease biomarkers search and for online surgery control. In this study, high resolution mass spectrometry analysis of eutopic and ectopic endometrium tissues (90 samples) is done using direct tissue spray mass spectrometry in both positive and negative ion modes. The most abundant peaks in the both ion modes are those corresponding to lipids. Species of three lipid classes are observed, phosphatidylcholines (PC), sphingomyelins (SM) and phosphoethanolamines (PE). Direct tissue analysis gives mainly information on PC and SM lipids (29 species) in positive ion mode and PC, SM and PE lipids (50 species) in negative ion mode which gives complementary data for endometriosis foci differentiation. The biggest differences were found for phospholipids with polyunsaturated acyls and alkils. Although, tissue spray shows itself as appropriate tool for tissue investigation, caution should be paid to the interpretation of mass spectra because of their higher complexity with more possible adducts formation and multiple interferences must be taken into account. The present work extends the application of direct tissue analysis for the rapid differentiation between endometriotic tissues of different foci.

The chemical sensitivity of urine metabolomics analysis is greatly compromised due to the large amounts of inorganic salts in urine (NaCl, KCl), which are detrimental to analytical instrumentation, e.g. chromatographic columns or mass spectrometers. Traditional desalting approaches applied to urine pretreatment suffer from the chemical losses, which reduce the information depth of analysis. We aimed to test a simple approach for the simultaneous preconcentration and desalting of organic solutes in urine based on the collection of induced bursting bubble aerosols above the surface of urine samples. Bursting bubbles were generated at ambient conditions by feeding gas through an air diffuser at the bottom of diluted (200 times in ultrapure water) urine solution (50–500 mL). Collected aerosols were analyzed by the direct-infusion electrospray ionization mass spectrometry (ESI–MS). The simultaneous preconcentration (ca. 6–12 fold) and desalting (ca. six–tenfold) of organic solutes in urine was achieved by the bursting bubble sample pretreatment, which allowed ca. three-times higher number of identified urine metabolites by high-resolution MS analysis. No chemical losses due to bubbling were observed. The increased degree of MS data clustering was demonstrated on the principal component analysis of data sets from the urine of healthy people and from the urine people with renal insufficiency. At least ten times higher sensitivity of trace drug detection in urine was demonstrated for clenbuterol and salbutamol. Our results indicate the high versatility of bubble bursting as a simple pretreatment approach to enhance the chemical depth and sensitivity of urine analysis. The approach could be attractive for personalized medicine as well as for the diagnostics of renal disorders of different etiology (diabetic nephropathy, chronic renal failure, transplant-associated complications, oncological disorders). Urine desalting and preconcentration in bursting bubbles.

Real-time feedback about dissected tissue during the neurosurgical procedure is strongly requested. A novel direct ionization mass spectrometric method for identifying pathological differences in tissues is proposed. The method is based on simultaneous extraction of tissue lipids and electrospray ionization which allows mass spectrometric data to be obtained directly from soft tissues. The advantage of this method is the stable flow of solvent, which leads to stable time-dependent spectra. The tissues included necrotized tissues and tumor tissues in different combinations. Capability for direct analysis of samples of dissected tissues during the neurosurgical procedure is demonstrated. Data validation is conducted by compound identification using precise masses from the MS profile, MS/MS, and isotopic distribution structure analysis. The method can be upgraded and applied for real-time identification of tissues during surgery. This paper describes the technique and its application perspective. For these purposes, other methods were compared with the investigated one and the results were shown to be reproducible. Differences in lipid profiles were observed even in tissues from one patient where distinctions between different samples could be poor. The paper presents a proof of concept for the method to be applied in neurosurgery particularly and in tissue analysis generically. The paper also contains preliminary results proving the possibility of observing differences in mass spectra of different tumors.

Here we report the observation that, depending on the solvent used for the electrospray, 2-nitrophloroglucinol undergoes a deprotonation from different sites forming two tautomeric gas phase ions. Those ions differ by the collision-induced dissociation (CID) spectra and by the gas phase hydrogen/deuterium (H/D) exchange kinetic. We performed H/D exchange in the electrospray ionization (ESI) source by saturation ESI region with vapors of deuterated solvent (D2O). It was observed that [2-nitrophloroglucinol-H]− exchanges two –OH hydrogens when MeOD is used as the spray solvent but when the spray solvent is 50:50 MeOD/D2O we observed an additional two H/D exchanges at the aromatic ring. We propose that the reaction occurs via a keto–enol tautomerization mechanism which was found to be energetically favorable.