Identificatie van plantaardige eiwitten in magere melkpoeder

In het kader van controle op gebruik van magere melkpoeder (MMP) in kunstmelkvoeder onderzoekt het RIKILT ca. 800 monsters per jaar. Er zijn aanwijzingen dat er plantaardige eiwitten en/of eiwithydrolysaten (bijvoorbeeld soja, tarwe, erwt, maïs, etc.) worden toegevoegd aan het subsidiabel MMP terwijl dat niet is toegestaan. Deze eiwitten zijn niet toegestaan en kunnen met de huidige analysetechnieken niet of in onvoldoende mate worden gedetecteerd. Voor het kunnen screenen en identificeren van plantaardige eiwitten in MMP zijn een aantal technieken gekozen om nader te onderzoeken in de tweede helft van 2005. Dit betreft immunoblotting, voorscheidingen d.m.v. voorextractie of HPLC, MALDI TOF MS, targeted LC-MS, untargeted LC-MS(/MS) en capillaire electrofores

IgE Cross-Reactivity of Cashew Nut Allergens

Background: Allergic sensitisation towards cashew nut often happens without a clear history of eating cashew nut. IgE cross-reactivity between cashew and pistachio nut is well described; however, the ability of cashew nut-specific IgE to cross-react to common tree nut species and other Anacardiaceae, like mango, pink peppercorn, or sumac is largely unknown. Objectives: Cashew nut allergic individuals may cross-react to foods that are phylogenetically related to cashew. We aimed to determine IgE cross-sensitisation and cross-reactivity profiles in cashew nut-sensitised subjects, towards botanically related proteins of other Anacardiaceae family members and related tree nut species. Method: Sera from children with a suspected cashew nut allergy (n = 56) were assessed for IgE sensitisation to common tree nuts, mango, pink peppercorn, and sumac using dot blo

Comparative LC-MS: A landscape of Peaks and Valleys

Quantitative proteomics approaches using stable isotopes are well-known and used in many labs nowadays. More recently, high resolution quantitative approaches are reported that rely on LC-MS quantitation of peptide concentrations by comparing peak intensities between multiple runs obtained by continuous detection in MS mode. Characteristic of these comparative LC-MS procedures is that they do not rely on the use of stable isotopes; therefore the procedure is often referred to as label-free LC-MS. In order to compare at comprehensive scale peak intensity data in multiple LC-MS datasets, dedicated software is required for detection, matching and alignment of peaks. The high accuracy in quantitative determination of peptide abundancies provides an impressive level of detail. This approach also requires an experimental set-up where quantitative aspects of protein extraction and reproducible separation conditions need to be well controlled. In this paper we will provide insight in the critical parameters that affect the quality of the results and list an overview of the most recent software packages that are available for this procedur

Regulation of the inductive phase of microspore embryogenesis in Brassica napus

In cultured microspores from Brassica napus, embryogenesis can be synchronously and irreversibly induced by elevating the culture temperature to 32°C for a minimum of 8 h. Culture at 18°C allows gametophytic development to continue, and results in the formation of pollen in vitro. This allows us to study the temperature controlled switch in developmental fate from gametophytic development to embryogenic development by molecular means. Analysis of protein synthetic patterns by [35S]-methionine incorporation and 2-dimensional gel electrophoresis, revealed that 25 proteins were differentially synthesized during the induction of microspore embryogenesis. Most of these proteins (17) appeared to belong to the class of heat shock proteins (HSPs). Four of these HSPs have been identified by Western blotting using antibodies raised against HSP17, HSP68 and HSP70. One protein that was only synthesized under embryogenic culture conditions, and did not belong to the heat inducible HSPs, is a candidate marker for early embryogenic development

Identification and characterization of digestive serine proteases from inhibitor-resistant Helicoverpa zea larval midgut

Protease inhibitors mediate a natural form of plant defence against insects, by interfering with the digestive system of the insect. In this paper, affinity chromatography was used to isolate trypsins and chymotrypsins from Helicoverpa zea larvae, which had been raised on inhibitor-containing diet. Sensitivity of the fractions to inhibition by plant proteinase inhibitors was tested, and compared to the sensitivity of proteinases found in insects raised on diet to which no inhibitor had been added. The isolated chymotrypsin activity was found to be less sensitive to plant protease inhibitors. The sensitivity of the isolated trypsin activity was found to be intermediate between completely sensitive trypsins and completely insensitive forms that have been previously described. Mass spectrometry was used to identify one trypsin and two chymotrypsins in the partially purified protease fraction. The sequence features of these proteases are discussed in relation to their sensitivity to inhibitors. The results provide insight in the enzymes deployed by Helicoverpa larvae to overcome plant defenc

Proteomic analysis of the major birch allergen Bet v 1 predicts allergenicity for 15 birch species

Pollen of the European and Asian white birch (Betula pendula and B. platyphylla) causes hay fever in humans. The allergenic potency of other birch species is largely unknown. To identify birch trees with a reduced allergenicity, we assessed the immunochemical characteristics of 15 species and two hybrids, representing four subgenera within the genus Betula, while focusing on the major pollen allergen Bet v 1. Antigenic and allergenic profiles of pollen extracts from these species were evaluated by SDS-PAGE and Western blot using pooled sera of birch-allergic individuals. Tryptic digests of the Bet v 1 bands were analyzed by LC-MSE to determine the abundance of various Bet v 1 isoforms. Bet v 1 was the most abundant pollen protein across all birch species. LC-MSE confirmed that pollen of all species contained a mixture of multiple Bet v 1 isoforms. Considerable differences in Bet v 1 isoform composition exist between birch species. However, isoforms that are predicted to have a high IgE-reactivity prevailed in pollen of all species. Immunoblotting confirmed that all pollen extracts were similar in immune-reactivity, implying that pollen of all birch species is likely to evoke strong allergic reactions