Oxana P. Trifonova

Blood plasma metabolites and the risk of developing lung cancer in Russia.

Lung cancer is one of the most common types of cancer in men, and is a leading cause of cancer-related deaths. Therefore, the identification of specific markers associated with a risk of lung cancer development, particularly metabolites that are more easily assayed, would be very valuable. To this end, а comparative metabolomics study of blood plasma samples collected from patients with lung cancer (n=100) and controls (n=100) recruited in Moscow was carried out. After the extraction of blood plasma proteins with methanol, the remaining plasma metabolite fractions were analyzed directly using mass spectrometry. Hundreds of cancer-associated metabolites were detected, and at least 70 metabolite ions with odds ratio values of 10–288 were found to be associated with the presence of cancer. Although these metabolites were present at higher levels in cancer patients, particularly in the early stages of disease, they did not correlate positively with cancer progression. On the basis of these findings, this metabolomics study of blood plasma samples from cancer patients shows that numerous cancer-associated metabolites were present in the studied population, and these could be used as factors for calculating the risk of lung cancer development in addition to currently used risk factors.

Diagnosing Impaired Glucose Tolerance Using Direct Infusion Mass Spectrometry of Blood Plasma

The goal of this study was to evaluate the capacity for mass spectrometry of blood plasma to diagnose impaired glucose tolerance (IGT). For this study, blood plasma samples from control subjects (n = 30) and patients with IGT (n = 20) were treated with methanol and low molecular weight fraction were then analyzed by direct infusion mass spectrometry. A total of 51 metabolite ions strongly associated with IGT were detected. The area under a receiver operating characteristic (ROC) curve (AUC) for diagnosing IGT that was based on an analysis of all these metabolites was 0.93 (accuracy 90%, specificity 90%, and sensitivity 90%). The associated reproducibility was 85%. The metabolites identified were also consistent with risk factors previously associated with the development of diabetes. Thus, direct infusion mass spectrometry of blood plasma metabolites represents a rapid, single-step, and reproducible method for the analysis of metabolites. Moreover, this method has the potential to serve as a prototype for clinical analyses that could replace the currently used glucose tolerance test with a more patient-friendly assay.

Application of 2-DE for studying the variation of blood proteome

2D electrophoresis (2-DE) is still a widely used proteomic method despite the availability of high-throughput approaches for protein identification. However, a literature survey only revealed a relatively small number of 2-DE-based studies of human blood proteome. We critically reviewed comparative 2-DE-based proteomic studies, in which groups of patients under examination involved more than ten individuals. Limitation on the number of samples is explained by the requirement to take into account the individual variation of blood proteome. The scanty amount of statistical data on quantitative variations of normal blood proteome may be one of the reasons for the poor applicability of proteomic biomarkers in clinical diagnostics.

Selection of the peptide mass tolerance value for protein identification with peptide mass fingerprinting

Peptide mass fingerprinting (PMF) is widely used for protein identification while studying proteome via time-of-flight mass spectrometer or via 1D or 2D electrophoresis. Peptide mass tolerance indicating the fit of theoretical peptide mass to an experimental one signifcantly influences protein identification. The role of peptide mass tolerance could be estimated by counting the number of correctly identified proteins for the reference set of mass spectra. The reference set of 400 Ultraflex (Bruker Daltonics, Germany) protein mass spectra was obtained for liver microsomes slices hydrolyzed via 1D gel electrophoresis. Using a Mascot server for protein identification, the peptide mass tolerance value varied within 0.02–0.40 Da with a step of 0.01 Da. The number of identified proteins changed up to 10 times depending on the tolerance. The maximal number of identified proteins was reported for the tolerance value of 0.15 Da (120 ppm) known to be 1.5–2-fold higher than the recommended values for such a type of mass spectrometer. The software program PMFScan was developed to obtain the dependence between the number of identified proteins and the tolerance values.

The proteomic study of Danio rerio embryos using one-dimensional electrophoresis and mass-spectrometry

Using one-dimensional proteomic mapping (combination of one-dimensional gel electrophoresis (1DE) with subsequent mass spectrometry MALDI-TOF-PMF) the protein profile of Danio rerio embryos has been investigated. The fish species Danio rerio is the most effective alternative model of vertebrates used for studies of drug toxicity (e.g. doxorubicin) due to its high degree of homology with human genome. The proteomic profiling resulted in identification of 84 proteins, including 15 vitellogenins. Using the procedure of preparation of homogenates of Danio rerio embryos optimized by ultrasonic treatment promoting removal of yolk basic proteins (vitellogenin) we have registered changes in the proteome profile of D. rerio embryos induced by doxorubicin (DOX). Growth D. rerio embryos in the medium with DOX caused the decrease in the number of vitellogenins, disappearance of cardiac troponins, and induction of caspase-3. All these observations are consistent with the literature data on doxorubicin-induced cardiotoxicity. The proposed method of 1D proteomic mapping may be used not only for protein identification but also for registration of changes in embryonic proteomic profile caused by drugs or any toxic compound for studying the mechanisms underlying induced toxicity.

Mass Spectrometry Analysis of Blood Plasma Lipidome as the Method of Disease Diagnostics, Evalution of Effectiveness and Optimization of Drug Therapy

A new method for the analysis of blood lipids based on direct mass spectrometry of lipophilic low molecular weight fraction of blood plasma has been considered. Such technique allows quantification of hundreds of various types of lipids and this changes existing concepts on diagnostics of lipid disorders and related diseases. Here we demonstrate the versatility and quickness of the method, which significantly simplify its wide use. This method is applicable for diagnostics of atherosclerosis, diabetes mellitus, cancer and other diseases. Detalization of plasma lipid composition at the molecular level by means of mass spectrometry allows to assess the effectiveness of therapy and to optimize the drug treatment of cardiovascular diseases by phospholipid preparations.