Oxidative protein labeling in mass-spectrometry-based proteomics

Oxidation of proteins and peptides is a common phenomenon, and can be employed as a labeling technique for mass-spectrometry-based proteomics. Nonspecific oxidative labeling methods can modify almost any amino acid residue in a protein or only surface-exposed regions. Specific agents may label reactive functional groups in amino acids, primarily cysteine, methionine, tyrosine, and tryptophan. Nonspecific radical intermediates (reactive oxygen, nitrogen, or halogen species) can be produced by chemical, photochemical, electrochemical, or enzymatic methods. More targeted oxidation can be achieved by chemical reagents but also by direct electrochemical oxidation, which opens the way to instrumental labeling methods. Oxidative labeling of amino acids in the context of liquid chromatography(LC)–mass spectrometry (MS) based proteomics allows for differential LC separation, improved MS ionization, and label-specific fragmentation and detection. Oxidation of proteins can create new reactive groups which are useful for secondary, more conventional derivatization reactions with, e.g., fluorescent labels. This review summarizes reactions of oxidizing agents with peptides and proteins, the corresponding methodologies and instrumentation, and the major, innovative applications of oxidative protein labeling described in selected literature from the last decade

Mutual Effect of <i>NAT2</i> rs1799930 (590G>A) Polymorphism and Alcohol Abuse on Risk of Acute Pancreatitis

Aim. Estimation of the contribution of rs1799930 (590G&gt;A) polymorphism of gene NAT2 to the development of acute alcoholic pancreatitis.Materials and methods. DNA samples were obtained from 547 unrelated patients with acute alcoholic pancreatitis and 573 unrelated individuals without gastrointestinal diseases. A survey selected individuals with the alcohol consumption of &gt;200 g/week pure ethanol two times a week or more during 10 or more years. Genotyping was performed with PCR using TaqMan allelic discrimination assays.Results. No association was observed between the NAT2 allelic rs1799930 (590G&gt;A) polymorphism, risk of acute alcoholic pancreatitis, duration and rate of alcohol consumption. The 590G&gt;A variant of rs1799930 in gene NAT2 correlated with an increased risk of acute alcoholic pancreatitis (odds ratio 2.16; 95% confidence interval 1.13–4.12) under alcohol consumption &gt;200 g/week pure ethanol.Conclusion. The rs1799930 G/A polymorphism of gene NAT2 increases the risk of acute pancreatitis under alcohol consumption &gt;200 g/week pure ethanol

The role of cytokine genetic polymorphism in development of acute pancreatitis: analysis of intergenic and environmental interactions

Aim of investigation. To study association interleukin-1 (IL-1) gene (rs16944), interleukin-6 (IL-6) gene (rs1800795), interleukin-10 (IL-10) gene (rs1800896) polymorphisms with development of acute pancreatitis (AP) in the Russian population. Material and methods. Whole blood samples were received from 297 AP patients and 238 healthy controls. Genotyping of IL-1 gene (rs16944) polymorphisms, IL-6 gene (rs1800795), IL-10 gene (rs1800896) was carried out by polymerase chain reaction with allele discrimination by TaqMan-probes. Results. The genetic polymorphism combination 511СТ×174GC of IL-1 and IL-6 genes was associated to high risk of AP development (OR=2.25, 95%-CI 1.45-3.49; p=0.0018). According to stratification analysis smoking patients with 511CT genotype had higher AP risk, then the patients with other genotypes (OR=2.22, 95%-CI 1.3-3.79; p=0.003). Paired combination of genotypes to disease risk analysis demonstrated that at 511СT×174GС genotype combination the AP risk is highest at alcohol abuse history for over 10 years (OR=2.88, 95%-CI 1.59-5.23; p=0.0004). Conclusion. Interleukin genetic polymorphism investigation may be useful at assessment of cytokine status in AP patients to predict the outcomes and to develop the personalized approach to treatment and prophylaxis

The role of polymorphism (rs1800566) of NAD(F)H quinone oxidoreductase-1 in development of acute pancreatitis

Aim of investigation. To estimate the role of genetic polymorphism (rs1800566) of NAD(F)H quinone oxidoreductase-1 in development of acute pancreatitis in the Russian population. Material and methods. Whole blood samples were received from 349 unrelated AP patients and 329 unrelated individuals of Russian nationality having no gastrointestinal diseases. Mean age of patients was 48.9±13.1 years, mean age of healthy controls - 47.8±12.1 years. Genotyping of NQO1 gene polymorphism (rs1800566) was carried out by real-time polymerase chain reaction. Results. The association of polymorphism (rs1800566) of NQO1 gene T/T genotype with high risk of AP development, mostly - acute and non-biliary pancreatitis, in males. The risk of disease was elevated in patients with TT genotype irrespective of intensity and duration of alcohol intake, as well as in smoking patients with ST genotype at alcohol consumption exceeding 10 years, even in those receiving less than 199 g of ethanol per week

The role of polymorphism (rs1800566) of NAD(F)H quinone oxidoreductase-1 in development of acute pancreatitis

Aim of investigation. To estimate the role of genetic polymorphism (rs1800566) of NAD(F)H quinone oxidoreductase-1 in development of acute pancreatitis in the Russian population. Material and methods. Whole blood samples were received from 349 unrelated AP patients and 329 unrelated individuals of Russian nationality having no gastrointestinal diseases. Mean age of patients was 48.9±13.1 years, mean age of healthy controls - 47.8±12.1 years. Genotyping of NQO1 gene polymorphism (rs1800566) was carried out by real-time polymerase chain reaction. Results. The association of polymorphism (rs1800566) of NQO1 gene T/T genotype with high risk of AP development, mostly - acute and non-biliary pancreatitis, in males. The risk of disease was elevated in patients with TT genotype irrespective of intensity and duration of alcohol intake, as well as in smoking patients with ST genotype at alcohol consumption exceeding 10 years, even in those receiving less than 199 g of ethanol per week

The Role of Phosphatidylethanolamine-N-methyltransferase (PEMT) Gene rs12449964 Polymorphism in the Development of Acute Pancreatitis and its Complications

Background. Acute pancreatitis is considered to be an important issue in modern medicine. The phosphatidylethanolamine-N-methyltransferase enzyme plays a significant role in the regulation of lipid metabolism by catalyzing the process of methylation of phosphatidylethanolamine to phosphatidylcholine. These lipids are key components of mitochondrial and cell membranes, providing their fluid and plastic properties and participating in the transport of fats, fatty acids and cholesterol. Along with its function in the synthesis of phosphatidylcholine, the methylation of phosphatidylethanolamine promotes the turnover of S-adenosylmethionine for the synthesis of cysteine and glutathione through transulphurisation. PEMT is a gene encoding the phosphatidylethanolamine-N-methyltransferase enzyme.Aim. To determine the role of PEMT C/T rs12449964 polymorphism in the risk of developing acute pancreatitis and its complications among Russian residents in Central Russia.Materials and methods. Whole blood samples were collected from 502 unrelated patients with acute non-biliary pancreatitis (97 women and 405 men) of Russian nationality who had been admitted to the surgical departments of the city of Kursk from 2015 to 2018, as well as from 513 unrelated individuals of Russian nationality without gastrointestinal diseases (101 women and 412 men). The average age of patients and healthy individuals was 48.9 ± 13.1 and 47.89 ± 12.1 years, respectively. Genomic DNA was isolated by a standard phenol-chloroform extraction method. Genotyping of rs12449964 polymorphism was performed using real-time PCR by allelic discrimination using a CFX96 Bio-Rad Laboratories amplifier (USA) with TaqMan probes and commercial TaqMan SNP Genotyping Assays reagents purchased from Applied Biosystems (USA).Results. The study has shown that the frequency of the C allele and the C/C PEMT C/T rs12449964 genotype was higher in the group of patients with acute pancreatitis, while the C/T genotype was predominant in the control group. C/T — T/T genotypes demonstrated a protective effect on the development of infected pancreatic necrosis, purulent necrotic peripancreatitis and severe acute pancreatitis.Conclusions. The disruption of phosphatidylethanolamine methylation processes increases the sensitivity of cells to oxidative stress, which can lead to the development of acute pancreatitis

Discrimination of Leucine and Isoleucine in Peptides Sequencing with Orbitrap Fusion Mass Spectrometer

An efficient approach to easy and reliable differentiation between isomeric leucine and isoleucine in peptide sequencing utilizes multistage electron transfer dissociation and higher energy collision activated dissociation in the Orbitrap Fusion mass spectrometer. The MS³ method involves production and isolation of primary odd-electron z^• ions, followed by radical site initiation of their fragmentation with formation of w-ions, characteristic of the isomeric amino acid residues. Six natural nontryptic peptides isolated from the secretion of frog <i>Rana ridibunda</i> were studied. Their lengths were in the range between 15 and 37 amino acids and the number of targeted isomeric (Leu/Ile) residues varied between 1 and 7. The experiments were successful in all 22 cases of Leu/Ile residues, leaving no doubts in identification. The method is extremely selective as the targeted w-ions appear to be the most intense in the spectra. The proposed approach may be incorporated into shotgun proteomics algorithms and allows for the development of an exclusively mass spectrometric method for automated complete <i>de novo</i> sequencing of various peptides and proteins

The Role of Phosphatidylethanolamine-N-methyltransferase (PEMT) Gene rs12449964 Polymorphism in the Development of Acute Pancreatitis and its Complications

Background. Acute pancreatitis is considered to be an important issue in modern medicine. The phosphatidylethanolamine-N-methyltransferase enzyme plays a significant role in the regulation of lipid metabolism by catalyzing the process of methylation of phosphatidylethanolamine to phosphatidylcholine. These lipids are key components of mitochondrial and cell membranes, providing their fluid and plastic properties and participating in the transport of fats, fatty acids and cholesterol. Along with its function in the synthesis of phosphatidylcholine, the methylation of phosphatidylethanolamine promotes the turnover of S-adenosylmethionine for the synthesis of cysteine and glutathione through transulphurisation. PEMT is a gene encoding the phosphatidylethanolamine-N-methyltransferase enzyme.Aim. To determine the role of PEMT C/T rs12449964 polymorphism in the risk of developing acute pancreatitis and its complications among Russian residents in Central Russia.Materials and methods. Whole blood samples were collected from 502 unrelated patients with acute non-biliary pancreatitis (97 women and 405 men) of Russian nationality who had been admitted to the surgical departments of the city of Kursk from 2015 to 2018, as well as from 513 unrelated individuals of Russian nationality without gastrointestinal diseases (101 women and 412 men). The average age of patients and healthy individuals was 48.9 ± 13.1 and 47.89 ± 12.1 years, respectively. Genomic DNA was isolated by a standard phenol-chloroform extraction method. Genotyping of rs12449964 polymorphism was performed using real-time PCR by allelic discrimination using a CFX96 Bio-Rad Laboratories amplifier (USA) with TaqMan probes and commercial TaqMan SNP Genotyping Assays reagents purchased from Applied Biosystems (USA).Results. The study has shown that the frequency of the C allele and the C/C PEMT C/T rs12449964 genotype was higher in the group of patients with acute pancreatitis, while the C/T genotype was predominant in the control group. C/T — T/T genotypes demonstrated a protective effect on the development of infected pancreatic necrosis, purulent necrotic peripancreatitis and severe acute pancreatitis.Conclusions. The disruption of phosphatidylethanolamine methylation processes increases the sensitivity of cells to oxidative stress, which can lead to the development of acute pancreatitis