Određivanje aminokiselinske sekvencije farmaceutskih peptida MALDI-TOF tandemnom spektrometrijom masa

Although the peptide ion mechanisms of pre-dissociation, dissociation and post-dissociation have been well-studied over the past fifteen years, their practical application still has to be implemented into modern mass spectrometry-driven proteomics and bioanalysis. Unambiguous peptide sequence determination by mass spectrometry relies on the idea that only one continuous series of ions in mass spectrum can assure full sequence determination and meets the requirements of peptide analysis quality. This set of rules defined for the peptide analysis by tandem mass spectrometry would generally improve an overall reliability and data accuracy. Based on the process mechanisms of gas-phase peptide bond dissociation, a relatively small and large model peptides are unambiguously analyzed (bivalirudin and exenatide) showing that derivatization concepts of the C- or N-terminus derivatization (SPITC and Lys-tag) can be avoided.Iako su mehanizmi preddisocijacije, disocijacije i postdisocijacije iona izučavani dugi niz godina, postoji još cijeli niz mehanizama raspada iona koji se mogu korisno upotrijebiti u modernoj spektrometriji masa, proteomici i bioanalizama. Nedvojbena aminokiselinska analiza spektrometrijom masa počiva na ideji da samo jedan i kontinuirani slijed iona u tandemnom spektru masa može potpuno potvrditi cjelovitu aminokiselinsku sekvenciju i na taj način zadovoljiti zahtjeve nedvojbene analize peptida. Niz pravila kojima se definira cjelovita i nedvojbena analiza peptida tandemnom spektrometrijom masa u konačnici može unaprijediti iščitavanje rezultata analize i pouzdanost dobivenih podataka. Na teorijskim zasadama disocijacije iona u plinskoj fazi u ovom je radu prikazana cjelovita analiza peptida. Na modelnim farmaceutskim peptidima bivalirudinu i eksenatidu pokazano je kako se i bez raširene upotrebe derivatizacijskih tehnika (SPITC i Lys-tag) može ostvariti nedvojbena analiza, iako se modifikacije cisteina akrilacijom, metilacijom ili karboksimetilacijom pri tome ne mogu izbjeći

Integrated dataset on acute phase protein response in chicken challenged with Escherichia coli lipopolysaccharide endotoxin

Data herein describe the quantitative changes in the plasma proteome in chickens challenged with lipopolysaccharide (LPS), a bacterial endotoxin known to stimulate the host innate immune system obtained by shotgun quantitative proteomic tandem mass tags approach using high-resolution Orbitrap technology. Statistical and bioinformatic analyses were performed to specify the effect of bacterial endotoxin. Plasma from chicken (N=6) challenged with Escherichia coli (LPS) (2 mg/kg body weight) was collected pre (0 h) and at 12, 24, 48, and 72 h post injection along with plasma from a control group (N=6) challenged with sterile saline. Protein identification and relative quantification were performed using Proteome Discoverer, and data were analysed using R. Gene Ontology terms were analysed by the Cytoscape application ClueGO based on Gallus gallus GO Biological Process database, and refined by REVIGO. Absolute quantification of several acute phase proteins, e.g. alpha-1-acid glycoprotein (AGP), serum amyloid A (SAA) and ovotrensferrin (OVT) was performed by immunoassays to validate the LC-MS results. The data contained within this article are directly related to our research article”Quantitative proteomics using tandem mass tags in relation to the acute phase protein response in chicken challenged with Escherichia coli lipopolysaccharide endotoxin” [1]. The raw mass spectrometric data generated in this study were deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD009399 (http://proteomecentral.proteomexchange.org/cgi/GetDataset?ID=PXD009399)

Sugar beet cells’ cellular and extracellular events taking place in response to drought and salinity

Salt and drought stress are important abiotic factors that negatively affect plant growth and yield. To understand how these stress factors affect metabolism at the cellular level, we analyzed cation concentrations and expression of cellular and extracellular proteins, as well as their functions and types. Cells of the industrially important halophyte sugar beet were exposed to 300 mM NaCl and 600 mM mannitol as stressors in modified Gamborg B5 liquid nutrient medium (PG0). Severe stress altered the intracellular concentrations of most of the measured cations. The cellular proteome revealed that both stressors provoked significant differential regulation of 110 cellular proteins. About 80% of the identified proteins were classified in metabolism, energy, or cell rescue, defense and virulence categories. We identified several novel proteins that respond to stress, including a member of the bZIP family of transcription factors, a member of the glycine-rich RNA-binding proteins, and the K+ channel beta subunit. Among extracellular proteins we found previously unreported stress-responsive proteins, a beta-xylosidase and an isoform of chitinase. The obtained results indicate that salt and drought stress disturbed the concentrations of cellular cations and affected the expression of cellular and extracellular proteins in sugar beet cells

Hyphenated techniques liquid chromatography-mass spectrometry: basic methodology and applicati ons

Tekućinska kromatografi ja (LC) osnovna je višenamjenska separacijska tehnika koja se primjenjuje u modernim biološkim znanosti ma i srodnim područjima molekularne biologije kao što su analiti čka ili preparati vna kemija. Za razliku od mnogih drugih separacijskih tehnika koje nisu pogodne za razdvajanje termički nestabilnih molekula (npr. plinska kromatografija, GC) ili onih koje se ne mogu direktno spregnuti (npr. izoelektrično fokusiranje, IEF), tekućinska kromatografija može uspješno poslužiti za razdvajanje širokog raspona molekula kao što su to polimeri, male molekule farmaceutika ili njihovih metabolita, kao i pepti da i proteina. Odabirom takve metode tekućinske kromatografi je koja ne šteti analizi spektrometrijom masa (hlapljivi puferi, stabilan i nizak protok, upotreba polarnih organskih otapala), tekućinski kromatograf može se jednostavno spregnuti sa spektrometrom masa. Izvor iona, analizator (ili kombinacija više analizatora u istom instrumentu, tzv. tandemska spektrometrija masa) i detektor iona odabiru se u ovisnosti o vrstama analiza, a koje pak mogu biti jednostavne analize određenog iona ili kvalitati vno substrukturalne i kvanti tati vne analize kompleksnih smjesa. Da bi se olakšalo određivanje strukture ili kvanti tati vna analiza, u proteklom desetljeću su razvijene različite programske aplikacije i pretraživači baza podataka.Liquid chromatography (LC) is a basic versati le separati on technique widely used in modern life sciences especially in the fi elds closely related to molecular biology, namely in the analytical or preparative chemistry. Unlike many other separati on techniques, which are unsuitable for thermally degradable molecules (e.g. gas chromatography, GC), or which are not feasible for on-line coupling (e.g. isoelectric focusing, IEF), liquid chromatography can efficiently separate a very wide range of large molecules like polymers, peptides and proteins as well as small molecules like drug or drug metabolites. Choosing the proper LC method that uti lize mass spectrometry (MS) friendly conditi ons (volati le buff ers, stable and low fl ow rate, polar organic solvents etc.), liquid chromatography system can be easily hyphenated to mass spectrometer. Ionizati on source, analyzer (or combinati on of more analyzers in one instrument, the so called tandem mass spectrometry) and ion detector are selected according to the analysis requirements that might range from simple one ion qualitative analysis to substructural qualitati ve, and quanti tati ve analysis of complex mixtures. To facilitate elucidati on of structure or quanti tati ve analysis different soft ware applicati ons and data bases search engines were developed during the last decade

Identification of possible new salivary biomarkers of stress in sheep using a high-resolution quantitative proteomic technique

The aim of this study was to identify biological pathways and proteins differentially expressed in the saliva proteome of sheep after the application of a model of stress, using high-resolution quantitative proteomics. In addition, one of the proteins differently expressed was verified and evaluated as a possible biomarker of stress in this species. Saliva paired samples from eight sheep before and after the application of a model of stress based on shearing were analysed using tandem mass tags (TMT). The TMT analysis allowed for the identification of new stress-related metabolic pathways and revealed 13 proteins, never described in saliva of sheep, that were differentially expressed between before and after the stress. Six of these proteins pertain to four major metabolic pathways affected, namely: canonical glycolysis, oxygen transport, neural nucleus development, and regulation of actin cytoskeleton reorganization. The rest of proteins were unmapped original proteins such as acyl-coenzyme-A-binding protein; complement C3; alpha-2-macroglobulin isoform-X1; type-II small proline-rich protein; lactoferrin; secretoglobin family-1D-member; and keratin, type-II cytoskeletal 6. Of these proteins, based on its biological significance and specific immunoassay availability, lactoferrin was selected for further validation. The immunoassay intra- and inter-assay coefficients of variation were lower than 13%. The method showed good linearity under dilution and recovery, and the detection limit was low enough to detect salivary lactoferrin levels. A significant decrease (P < 0.01) in salivary lactoferrin concentration in the sheep following the application of the model of stress was observed, suggesting that this protein could be a potential salivary biomarker of stress situations in sheep

Novel biomarkers in cats with congestive heart failure due to primary cardiomyopathy

The pathogenesis of feline cardiomyopathy and congestive heart failure (CHF) requires further understanding. In this study, we assessed serum proteome change in feline CHF, aiming to identify novel biomarker for both research and clinical use. The study comprised 15 cats in CHF, 5 cats in preclinical cardiomyopathy and 15 cats as healthy controls. Serum proteome profiles were obtained by tandem mass tag labelling followed by mass spectrometry. Protein concentrations in CHF cats were compared with healthy controls. Western blot was performed for proteomic validation. Correlations were assessed between the altered proteins in CHF and clinical variables in cats with cardiomyopathy to evaluate protein-cardiac association. Bioinformatic analysis was employed to identify pathophysiological pathways involved in feline CHF. Sixteen serum proteins were significantly different between CHF and healthy control cats (P < .05). These included serine protease inhibitors, apolipoproteins and other proteins associated with inflammation and coagulation. Clinical parameters from cats with cardiomyopathy significantly correlated with the altered proteins (P < .05). Bioinformatic analysis identified 13 most relevant functional profiles in feline CHF, which mostly associated with extracellular matrix organization and metabolism. Data are available via ProteomeXchange with identifier PXD017761

The plasma proteome and the acute phase protein response in canine pyometra

Canine pyometra is a common inflammatory disease of uterus in sexually mature bitches caused by secondary bacterial infection, leading to change in plasma proteins associated with the innate immune system. Proteomic investigation is increasingly being applied to canine diseases in order to identify and quantify significant changes in the plasma proteome. The aim of the study was to assess and quantify changes in plasma proteome profiles of healthy and pyometra affected bitches using a TMT-based high-resolution quantitative proteomic approach. As a result, 22 proteins were significantly down-regulated including transthyretin, antithrombin III, retinol-binding protein, vitamin D binding protein, paraoxonase 1, and kallikrein, while 16 were significantly up-regulated including haptoglobin light chain, alpha-1-acid glycoprotein, C-reactive protein precursor, and lipopolysaccharide-binding protein in dogs with pyometra. Pathway analysis indicated that acute inflammatory response, regulation of body fluid levels, protein activation cascade, the humoral immune response, and phagocytosis were affected in pyometra. Validation of biological relevance of the proteomic study was evident with significant increases in the concentrations of haptoglobin, C-reactive protein, alpha 1 acid glycoprotein, and ceruloplasmin by immunoassay. Pyometra in bitches was shown to stimulate an increase in host defence system proteins in response to inflammatory disease including the acute phase proteins

Effect of Tff3 Deficiency and ER Stress in the Liver

Endoplasmic reticulum (ER) stress, a cellular condition caused by the accumulation of unfolded proteins inside the ER, has been recognized as a major pathological mechanism in a variety of conditions, including cancer, metabolic and neurodegenerative diseases. Trefoil factor family (TFFs) peptides are present in different epithelial organs, blood supply, neural tissues, as well as in the liver, and their deficiency has been linked to the ER function. Complete ablation of Tff3 expression is observed in steatosis, and as the most prominent change in the early phase of diabetes in multigenic mouse models of diabesity. To elucidate the role of Tff3 deficiency on different pathologically relevant pathways, we have developed a new congenic mouse model Tff3-/-/C57BL6/N from a mixed background strain (C57BL6/N /SV129) by using a speed congenics approach. Acute ER stress was evoked by tunicamycin treatment, and mice were sacrificed after 24 h. Afterwards the effect of Tff3 deficiency was evaluated with regard to the expression of relevant oxidative and ER stress genes, relevant proinflammatory cytokines/chemokines, and the global protein content. The most dramatic change was noticed at the level of inflammation-related genes, while markers for unfolded protein response were not significantly affected. Ultrastructural analysis confirmed that the size of lipid vacuoles was affected as well. Since the liver acts as an important metabolic and immunological organ, the influence of Tff3 deficiency and physiological function possibly reflects on the whole organism