Optimization of the linear quantification range of an online trypsin digestion coupled liquid chromatography–tandem mass spectrometry (LC–MS/MS) platform

Tandem mass spectrometry (MS/MS)-based proteomic workflows with a bottom-up approach require enzymatic digestion of proteins to peptide analytes, usually by trypsin. Online coupling of trypsin digestion of proteins, using an immobilized enzyme reactor (IMER), with liquid chromatography (LC) and MS/MS is becoming a frequently used approach. However, finding IMER digestion conditions that allow quantitative analysis of multiple proteins with wide range of endogenous concentration requires optimization of multiple interactive parameters: digestion buffer flow rate, injection volume, sample dilution, and surfactant type/concentration. In this report, we present a design of experiment approach for the optimization of an integrated IMER-LC–MS/MS platform. With bovine serum albumin as a model protein, the digestion efficacy and digestion rate were monitored based on LC–MS/MS peak area count versus protein concentration regression. The optimal parameters were determined through multivariate surface response modeling and consideration of diffusion controlled immobilized enzyme kinetics. The results may provide guidance to other users for the development of quantitative IMER-LC–MS/MS methods for other proteins.     

Functional properties of air-classified yellow pea (Pisum sativum)fractions

The objective of the present investigation was the characterisation of yellow pea (Pisum sativum L) fractions obtained on a commercial scale by air classification of the milled peas. Some analytical aspects of the process were investigated including particle size distribution of fractions and their functional properties.     

Automated solid-phase extraction and measurement of perfluorinated organic acids and amides in human serum and milk

Organic fluorochemicals are used in multiple commercial applications including surfactants, lubricants, paints, polishes, food packaging, and fire-retarding foams. Recent scientific findings suggest that several perfluorochemicals (PFCs), a group of organic fluorochemicals, are ubiquitous contaminants in humans and animals world wide. Furthermore, concern has increased about the toxicity of these compounds. Therefore, monitoring human exposure to PFCs is important. We have developed a high-throughput method for measuring trace levels of 13 PFCs (2 perfluorosulfonates, 8 perfluorocarboxylates, and 3 perfluorosulfonamides) in serum and milk using an automated solid-phase extraction (SPE) cleanup followed by high-performance liquid chromatography-tandem mass spectrometry. The method is sensitive, with limits of detection between 0.1 and 1 ng in 1 mL of serum or milk, is not labor intensive, involves minimal manual sample preparation, and uses a commercially available automated SPE system. Our method is suitable for large epidemiologic studies to assess exposure to PFCs. We measured the serum levels of these 13 PFCs in 20 adults nonoccupationally exposed to these compounds. Nine of the PFCs were detected in at least 75% of the subjects. Perfluorooctanesulfonate (PFOS), perfluorohexanesulfonate (PFHxS), 2-(N-methylperfluorooctane-sulfonamido)acetate (Me-PFOSA-AcOH), perfluorooctanoate (PFOA), and perfluorononanoate (PFNA) were found in all of the samples. The concentration order and measured levels of PFOS, PFOA, Me-PFOSA-AcOH, and PFHxS compared well with human serum levels previously reported. Although no human data are available for the perfluorocarboxylates (except PFOA), the high frequency of detection of PFNA and other carboxylates in our study suggests that human exposure to long-alkyl-chain perfluorocarboxylates may be widespread. We also found PFOS in the serum and milk of rats administered PFOS by gavage, but not in the milk of rats not dosed with PFOS. Furthermore, we did not detect most PFCs in two human milk samples. These findings suggest that PFCs may not be as prevalent in human milk as they are in serum. Additional studies are needed to determine whether environmental exposure to PFCs can result in PFCs partitioning into milk. Large epidemiological studies to determine the levels of PFCs among the U.S. general population are warranted.     

Investigation of the Antihypertrophic and Antifibrotic Effects of Losartan in a Rat Model of Radiation-Induced Heart Disease

Radiation-induced heart disease (RIHD) is a potential late side-effect of thoracic radiotherapy resulting in left ventricular hypertrophy (LVH) and fibrosis due to a complex pathomechanism leading to heart failure. Angiotensin-II receptor blockers (ARBs), including losartan, are frequently used to control heart failure of various etiologies. Preclinical evidence is lacking on the anti-remodeling effects of ARBs in RIHD, while the results of clinical studies are controversial. We aimed at investigating the effects of losartan in a rat model of RIHD. Male Sprague-Dawley rats were studied in three groups: (1) control, (2) radiotherapy (RT) only, (3) RT treated with losartan (per os 10 mg/kg/day), and were followed for 1, 3, or 15 weeks. At 15 weeks post-irradiation, losartan alleviated the echocardiographic and histological signs of LVH and fibrosis and reduced the overexpression of chymase, connective tissue growth factor, and transforming growth factor-beta in the myocardium measured by qPCR; likewise, the level of the SMAD2/3 protein determined by Western blot decreased. In both RT groups, the pro-survival phospho-AKT/AKT and the phospho-ERK1,2/ERK1,2 ratios were increased at week 15. The antiremodeling effects of losartan seem to be associated with the repression of chymase and several elements of the TGF-β/SMAD signaling pathway in our RIHD model.     

Somatostatin Primes Endothelial Cells for Agonist-Induced Hyperpermeability and Angiogenesis In Vitro

Somatostatin is an inhibitory peptide, which regulates the release of several hormones, and affects neurotransmission and cell proliferation via its five G_i protein-coupled receptors (SST_1-5). Although its endocrine regulatory and anti-tumour effects have been thoroughly studied, little is known about its effect on the vascular system. The aim of the present study was to analyse the effects and potential mechanisms of somatostatin on endothelial barrier function. Cultured human umbilical vein endothelial cells (HUVECs) express mainly SST₁ and SST₅ receptors. Somatostatin did not affect the basal HUVEC permeability, but primed HUVEC monolayers for thrombin-induced hyperpermeability. Western blot data demonstrated that somatostatin activated the phosphoinositide 3-kinases (PI3K)/protein kinase B (Akt) and p42/44 mitogen-activated protein kinase (MAPK) pathways by phosphorylation. The HUVEC barrier destabilizing effects were abrogated by pre-treating HUVECs with mitogen-activated protein kinase kinase/extracellular signal regulated kinase (MEK/ERK), but not the Akt inhibitor. Moreover, somatostatin pre-treatment amplified vascular endothelial growth factor (VEGF)-induced angiogenesis (3D spheroid formation) in HUVECs. In conclusion, the data demonstrate that HUVECs under quiescence conditions express SST₁ and SST₅ receptors. Moreover, somatostatin primes HUVECs for thrombin-induced hyperpermeability mainly via the activation of MEK/ERK signalling and promotes HUVEC proliferation and angiogenesis in vitro.     

The effect of pre-treatment on the anthocyanin and flavonol content of black currant juice (Ribes nigrum L.) in concentration by reverse osmosis

Reverse osmosis process for the concentration of black currant juice was carried using AFC-99 tubular membrane at 30 °C and 45 bar. The contents of selected flavonols and anthocyanins were analyzed after centrifugation; enzyme treatment by Panzym Super E and by Rohapect berry followed by centrifugation; and ultrafiltration black currant juices and juice concentrates. The total soluble solid (TSS) content of the juices increased from the initial 17.6–17.9 °Brix to 24–24.8 °Brix in the case of the centrifuged juice in the concentration process. Similarly, it increased from 14.5–15.5 °Brix to 23.1–23.4 °Brix for the Panzym Super E treated juice, and from 16.1–16.9 °Brix to 22.5–23.1 °Brix for the Rohapect berry treated black currant juices. The ultrafiltered juice had the lowest initial TSS content between 14.1 and 14.9 °Brix and it increased to 22.1–23.1 °Brix. The average permeate fluxes during the concentration process were 7.3 L m⁻² h⁻¹ for the centrifuged juice, 11.9 L m⁻² h⁻¹ for the Panzym Super E treated juice, 9.2 and 13.1 L m⁻² h⁻¹ for the Rohapect berry treated and ultrafiltered juice, respectively. Analysis indicated that the enzymatic treatment resulted in the increase of anthocyanin and flavonol content of the juices. The centrifugation process decreased the amount of anthocyanins and flavonols to some extent. The juice clarified by ultrafiltration had significantly lower concentrations of anthocyanins and flavonols, while the juices treated by Panzym Super E had the highest levels of these flavonoids. This study recommends enzymatic pre-treatment by Panzym Super E, since it improves the permeate flux in reverse osmosis during the concentration process, and results in a juice concentrates highest in anthocyanins and flavonols.     

Factor XIII B Subunit Polymorphisms and the Risk of Coronary Artery Disease

The aim of the case-control study was to explore the effect of coagulation factor XIII (FXIII) B subunit (FXIII-B) polymorphisms on the risk of coronary artery disease, and on FXIII levels. In the study, 687 patients admitted for coronary angiography to investigate suspected coronary artery disease and 994 individuals representing the Hungarian population were enrolled. The patients were classified according to the presence of significant coronary atherosclerosis (CAS) and history of myocardial infarction (MI). The F13B gene was genotyped for p.His95Arg and for intron K nt29756 C>G polymorphisms; the latter results in the replacement of 10 C-terminal amino acids by 25 novel amino acids. The p.His95Arg polymorphism did not influence the risk of CAS or MI. The FXIII-B intron K nt29756 G allele provided significant protection against CAS and MI in patients with a fibrinogen level in the upper tertile. However, this effect prevailed only in the presence of the FXIII-A Leu34 allele, and a synergism between the two polymorphisms was revealed. Carriers of the intron K nt29756 G allele had significantly lower FXIII levels, and FXIII levels in the lower tertile provided significant protection against MI. It is suggested that the protective effect of the combined polymorphisms is related to decreased FXIII levels.