The use of tryptic food protein digests data in public proteomic repositories to assess the effects of chemical and post-translational modifications on digestion outcomes

Porcine-derived trypsin generated proteomic data of the major peanut allergen Ara h 1 from the peanut was reassessed to search for possible facilitating/hindrance effects on trypsin digestion efficacy caused by post-translational and chemical modifications (PTMs) positioned on arginine or lysine (K/R) residues. If the potential effects caused by PTMs are observed with porcine trypsin, they can be just augmented and more pronounced within human intestinal digestion. The reasoning is in inferior performance of human trypsin compared to porcine-derived used in proteomic digestion protocols, also in the lower trypsin-to-sample ratio and much shorter digestion times, even though gastric digestion precedes and trypsin is not the sole digestive enzyme. A novel method was developed to decipher cleavage or miscleavage outcomes at scissile bonds in each, modified and unmodified sequence counterparts, using PEAKS Studio-X+ (Bioinformatics Solutions Inc., Ontario, Canada) in the reassessment of high-resolution tandem mass spectrometry data, from 18-hour long trypsin digestion proteomic protocols. In general, eight site-specific and modified K/R residues with methylation, dihydroxy and formylation showed significantly higher content of miscleaved bonds (at least >10%) compared to their unmodified counterpart peptides. Specifically, dihydroxylation and formylation hindered trypsin efficacy, while methylation on several K/R showed opposite effects. It is essential to elucidate the specific impacts of modifications on trypsin digestion performance and if there are additional effects generated by food processing, which could influence digestion outcomes and allergenicity of food proteins/peptides.Book of abstract: 4th Belgrade Bioinformatics Conference, June 19-23, 202

Trypsin as a proteomic probe to assess food protein digestibility in relation to post- translational modifications

Background: We have undertaken study on our porcine-derived trypsin generated proteomic data of the major peanut allergen Ara h 1 from the raw and roasted peanut, to assess possible facilitating/hindrance effects on trypsin digestion efficacy caused by post- translational and chemical modifications (PTMs) positioned on K/R residues. If potential hindrance effects caused by PTMs are observed with porcine trypsin, then they can be just augmented and more pronounced within human intestinal digestion. The logic for such reasoning is in inferior performance of human trypsin compared to porcine-derived used in proteomic digestion protocols, also the lower trypsin-to-sample ratio and much shorter digestion times, even though gastric digestion precedes and trypsin is not the sole digestive enzyme. Methods: Novel method was developed to decipher outcomes at scissile bonds using PEAKS Studio-X+ in reassessment of high-resolution tandem mass spectrometry data on 18h-long trypsin digestion protocol. Results: In eight modified K/R residues involving methylation, dihydroxy and formylation, differences in extent of miscleavage between modified and unmodified peptides, were significantly higher (>10%) in modified peptides. Conclusion: It is important to elucidate impact of modifications on trypsin digestion performance, but also on other proteases involved in digestion process due to possible effects on allergenicity of food proteins/peptides.Related to Book of Abstracts: [https://www.cost-infogest.eu/content/download/4051/35805/file/V-ICFD%20Book%20of%20Abstracts.pdf]Related to record of lecture: [https://www.youtube.com/watch?v=Dj0_1fyY724

Methods Development for Protein and Modifications Profiling

Post-translational modifications (PTMs) occur in many forms, and broadly influence protein behavior. High-resolution tandem mass spectrometry coupled with engines for the identification of unspecified PTMs, is a first-choice method for their global mapping. The significance of untargeted, in-depth PTM profiling of various proteomes and its method development, just emerged, in contrast to proteomic studies dealing with few selected static and dynamic modifications. In 2018, we have finalized our first study of comprehensive analysis of multiple polluted and environmentally preserved Timothy grass pollen samples that included novel method of relative quantification of proteins expressions and of open, quantitative PTM profiling. It was among the first ones that has appeared in the proteomics field. In addition to this, relative quantitative PTMs profiling of the raw and roasted peanut allergens was completed in 2020, including validation of the method by specific antibodies. This peanut PTM study was inspiration for the idea that porcine trypsin used in proteomic experiments can serve as a probe to decipher differences in scissile bond hydrolysis caused by PTMs, with the steric and/or charge changes causing possible hindrance or facilitation to its activity. We further hypothesized that effects observed would be even more pronounced with human trypsin, since it is less efficient compared to the porcine counterpart. Finally, we developed dual manual method to reassess our data of the major peanut allergen Ara h 1 from the raw and roasted peanut, to confirm or rule out the first hypothesis (do PTMs positioned on lysine/arginine residues facilitate or hinder porcine trypsin digestion efficacy). We believe that this topic is relevant from the aspect of human gastrointestinal digestion of food allergens and PTMs introduced by food processing. For further advancement in this area, novel algorithms based on deep machine learning, capable of reporting cleavage and miscleavage events separately within unmodified and modified part of protein sequences are warranted

Protein modifications screening of raw and thermally treated meat gastrointestinal digesta

Meat samples were subjected to thermal processing combined with simulated INFOGEST in vitro gastrointestinal (GI) digestion. Protein modifications (PMs) were screened with commercially available PM-specific antibodies. Specific proteins at 20, 37, 50, and 65 kDa react to more than 3 PM-specific antibodies among meat proteins. Lysine methylation and methionine oxidation were the most prominent PMs in WB. Mass spectrometry confirmed bands at ≈20 kDa as allergenic proteins: sarcoplasmic calcium-binding protein in oyster, 37 kDa as tropomyosin in shrimp, oyster, and abalone. MS-identified PMs of shellfish allergens were aligned to the IgE binding epitopes. GI digestion-resistant peptides of shellfish proteins were identified as paramyosins in oyster and abalone and SBP in shrimp. Our results point to the high susceptibility of immunodominant epitopes of major shellfish allergens to PMs. In TPM, saturation of oxidative modification increases with thermal processing resulting in higher susceptibility of TPM to gastric digestion

Comparative quantitative immunoproteomic study of raw and roasted peanut major allergen modifications

Peanut allergy affects approximately up to 3 % of children and up to 2 % of the adult world population, causing reactions ranging from mild to severe. Major peanut allergens are well characterized but little is known about their post-translational modifications and even less is known about the influence of thermal treatment on their profile. Protein post-translational modification patterns may differ between raw and thermally treated peanuts, which could affect its functional properties, such as allergic potential. In this study we combined proteomic and immunological methods to characterize the modifications or proteoforms of four major peanut allergens - Ara h 1, Ara h 2, Ara h 3 and Ara h 6 in raw and roasted peanut. Bottom-up high-resolution accurate mass spectrometry and a specialized proteomics software package to identify, map and compare modifications of major peanut allergens between differently treated peanut kernels. Modification-specific antibody western blot was used to confirm the presence of modifications on major allergens in both extracts. Twenty different post-translational modifications in four prominent peanut allergens (Ara h 1-3, 6) were identified, while twelve were quantitatively compared between raw and roasted peanuts by high-resolution mass spectrometry and a proprietary proteomics software. post-translational modification specific antibodies confirmed the presence of these modifications in western-blots of raw and roasted peanuts. This study initiates appreciation of modifications and thermal processing affecting food quality, and development of state-of-the-art methodology in the risk assessment of allergen contamination.Book of Abstract

Optimization of expression, purification and HRMS characterization of recombinant N-protein fragment from SARS-CoV-2

Nucleocapsid (N) protein is the most abundant SARS-CoV-2 virus derived protein and strong immunogen which can be used as a component of the immunological tests for the diagnosis of SARS-CoV-2 infection. Recombinant fragment of N-protein (58–419 aa) was expressed in E. coli in a soluble form using developed optimized protocol of expression (16-18h, 37 °C, 0.4 mM IPTG). After lysis of cells, N-protein from soluble fraction of lysate was purified using optimized protocol for purification by immobilized metal affinity chromatography on Ni-Sepharose in two repeated steps under different elution conditions. Obtained fraction of N-protein after the second chromatography was desalted and concentrated using phosphate buffer solution and ultrafiltration. The purity of isolated N-protein was deterrmined by SDS PAGE, while high resolution mass spectrometry was used for its characterization. Isolated N-protein was over 90% purity and identified as the most intense and abundant protein fragment, with PEAKS PTM score of 508 and sequence coverage of over 70%, including 173 unique peptides

Zajednički utjecaj ključnih onečišćivača zraka na težinu COVID-a 19 – predviđanje zasnovano na analizi toksikogenomičkih podataka

Considering that some researchers point to a possible influence of air pollution on COVID-19 transmission, severity, and death rate, the aim of our in silico study was to determine the relationship between the key air pollutants [sulphur dioxide (SO2), carbon monoxide (CO), particulate matter (PMx), nitrogen dioxide (NO2), and ozone (O3)] and COVID-19 complications using the publicly available toxicogenomic analytical and prediction tools: (i) Comparative Toxicogenomic Database (CTD) to identify genes common to air pollutants and COVID-19 complications; (ii) GeneMANIA to construct a network of these common and related genes; (iii) ToppGene Suite to extract the most important biological processes and molecular pathways; and (iv) DisGeNET to search for the top gene-disease pairs. SO2, CO, PMx, NO2, and O3 interacted with 6, 6, 18, 9, and 12 COVID-19-related genes, respectively. Four of these are common for all pollutants (IL10, IL6, IL1B, and TNF) and participate in most (77.64 %) physical interactions. Further analysis pointed to cytokine binding and cytokine-mediated signalling pathway as the most important molecular function and biological process, respectively. Other molecular functions and biologica processes are mostly related to cytokine activity and inflammation, which might be connected to the cytokine storm and resulting COVID-19 complications. The final step singled out the link between the CEBPA gene and acute myelocytic leukaemia and between TNFRSF1A and TNF receptor-associated periodic fever syndrome. This indicates possible complications in COVID-19 patients suffering from these diseases, especially those living in urban areas with poor air quality.COVID-19 (engl. coronavirus disease 2019) respiratorna je bolest prouzročena infekcijom SARS-CoV-2 virusom (engl. severe acute respiratory syndrome coronavirus 2). Pretpostavlja se da postoji utjecaj atmosferskih čimbenika, uključujući i onečišćenje zraka, na prenošenje koronavirusa, njegovu težinu i stopu smrtnosti. Stoga je cilj ovoga in silico istraživanja bio utvrditi odnos između ključnih onečišćivača zraka [sumporova dioksida (SO2), ugljikova monoksida (CO), lebdećih čestica (PMx), dušikova dioksida (NO2), ozona (O3)] i komplikacija COVID-a 19 korištenjem: (i) komparativne toksikogenomičke baze podataka (engl. Comparative Toxicogenomic Database, CTD) za dobivanje gena, međusobno povezanih s onečišćivačima zraka i komplikacijama COVID-a 19, (ii) GeneMANIA servera za konstruiranje mreže između dobivenih I srodnih gena, (iii) ToppGene Suite za izdvajanje najvažnijih bioloških procesa/molekularnih puteva i (iv) DisGeNET baze podataka za traženje najvažnijih parova gen-bolest. Za SO2, CO, PMx, NO2 odnosno O3 utvrđena je interakcija sa 6, 6, 18, 9, odnosno 12 gena povezanih s komplikacijama COVID-a 19. Četiri su zajednička (IL10, IL6, IL1B i TNF) i u najvećem postotku (77,64 %) sudjeluju u fizičkim interakcijama. Vezivanje citokina i signalni put posredovan citokinima izdvojeni su kao najvažnija molekularna funkcija i biološki proces. Druge molekularne funkcije i biološki procesi uglavnom su bili povezani s aktivnošću citokina i upalom, što bi se moglo dovesti u vezu s citokinskom olujom i posljedičnim komplikacijama COVID-a 19. Utvrđena je veza između različitih bolesti i ispitivanih gena, posebice između CEBPA i akutne mijelogene leukemije (AML) te između TNFRSF1A i sindroma periodične vrućice povezanoga s TNF receptorom. To upozorava na moguće komplikacije u osoba zaraženih koronavirusom koje boluju od tih bolesti, poglavito kada su dodatno potaknute poremećajem funkcije spomenutih gena

Uzročnici akutnih trovanja sa smrtnim ishodom u Republici Srbiji u periodu od 2010. do 2018. godine

The aim of this investigation was to present data analysis on acute poisonings with fatal outcome in the Republic of Serbia in from 2010 to 2018, based on the published Annual reports of the National Poison Control Center of the Military Medical Academy (1). In the observed 9-year period, the most common cause of acute poisoning was alcohol (50%), followed by drugs (30.1%), while in the third and fourth place were psychoactive substances (7.1%) and gases 4%. Corrosive and pesticide poisoning was present in only 2% of the total number of examined patients per year. The most common reason for the hospitalization was due to drug poisoning (71%), corrosives (9%), pesticides (5%), psychoactive substances (4%), gases (4%) and other pathogens (7%). From 2010 to 2018, number of deaths due to acute poisoning ranged from 26 to 43 annually with an average value of 33. Of 290 deceased patients, 101 (34.9%) were of drug poisoning, 108 (38.5%) corrosives, 28 (9.8%) pesticides, 4 (1.4%) gases, 14 (4.6%) psychoactive substances and 37 (12.4%) other substances, predominantly alcohols and mushrooms. Combination of two or more drugs most often led to death (n=51), while among corrosives, hydrochloric acid was the most common (n=40). Of the pesticides, the most common were organophosphorus insecticides (n=14), while heroin was the most common psychoactive substance (n=7). The analyzed data indicated the importance of preventive measures that need to be carried out in our country to reduce the number of acute poisonings, especially the most severe forms that lead to death.Cilj ovog rada bio je da se prikaže analiza podataka o akutnim trovanjima sa smrtnim ishodom u Republici Srbiji u periodu od 2010. do 2018. godine na osnovu publikovanih Godišnjaka Nacionalanog centra za kontrolu trovanja Vojnomedicinske akademije (1). U posmatranom devetogodišnjem periodu najčešći uzročnik akutnih trovanja je alkohol (50%), zatim lekovi (30,1%), dok su na trećem i četvrtom mestu psihoaktivne supstance (7,1%) i gasovi 4%. Samo oko 2% od ukupnog broja pregledanih pacijenata godišnje su slučajevi trovanja korozivima i pesticidima. Najčešći razlog hospitalizacije je usled trovanja lekovima (71%), zatim korozivima (9%), pesticidima (5%), psihoaktivnim supstancama (4%), gasovima (4%) i drugim uzročnicima (7%). Od 2010. do 2018. godine broj letalnih ishoda usled akutnog trovanja se kretao od 26 do 43 slučaja godišnje sa prosečnom vrednošću od 33 slučaja po godini. Od 290 preminula pacijenta, 101 (34,9%) slučaj je bilo trovanje lekovima, 108 (38,5%) korozivima, 28 (9,8%) pesticidima, 4 (1,3%) gasovima, 14 (4,6%) psihoaktivnim supstancama i 37 (12,4%) ostalim supstancama, dominantno alkoholima i gljivama. Kombinacija dva i više lekova je najčešće dovela do smrtnog ishoda (n=51), dok je među korozivima, najzastupljenija bila hlorovodonična kiselina (n=40). Od pesticida kao uzročnika akutnih trovanja sa smrtnim ishodom najzastupljeniji su organofosforni insekticidi (n=14), a prema broju smrtnih ishoda od psihoaktivnih supstanci najviše je zastupljen heroin (n=7). Analizirani podaci ukazuju na značaj preventivnih aktivnosti koje je u našoj zemlji potrebno sprovesti kako bi se smanjio broj akutnih trovanja, a time i onih najtežih oblika koji dovode do smrtnog ishoda.VIII Kongres farmaceuta Srbije sa međunarodnim učešćem, 12-15.10.2022. Beogra

Expression, purification and immunological characterization of recombinant protein fragment from SARS-CoV-2

Serological testing is important method for diagnosis of severe acute respiratory syndrome coronavirus 2 (SARSCoV-2) infection. Nucleocapsid (N) protein is the most abundant virus derived protein and strong immunogen. We aimed to find its efficient, low-cost production, suitable for serological diagnosis. SARS-CoV-2 recombinant fragment of nucleocapsid protein (rfNP; 58–419 aa) was expressed in E. coli in soluble form, purified by immobilized metal ion affinity chromatography and strong cation exchange chromatography after which it was analyzed by Mass and CD spectrometry and characterized biochemically and immunologically. Purified rfNP has secondary structure of full-length recombinant N protein, with high percentage of disordered structure (34.2%) and of β-sheet (40.7%). rfNP was tested in immunoblot using sera of COVID-19 convalescent patients. ELISA was optimized with sera of RT-PCR confirmed positive symptomatic patients and healthy individuals. IgG detection sensitivity was 96% (47/50) and specificity 97% (67/68), while IgM detection was slightly lower (94% and 96.5%, respectively). Cost-effective approach for soluble recombinant N protein fragment production was developed, with reliable IgG and IgM antibodies detection of SARS-CoV-2 infection.Book of Abstract