Nanoflow-HPLC-tandem mass spectrometry (MS/MS) was used to analyze the peptide fraction of breast milk samples collected from a single non-atopic donor on different days (10 samples) after receiving an oral load of cow's milk (by drinking 200 mL of bovine milk). In addition, breast milk was sampled from the same lactating mother over a 6-h period at five time points after drinking cow's milk. We aimed to trace the intra-individual variability and to define a time profile of the excretion of dietary peptides into breast milk. Overall, 21 peptides exclusively originating from both bovine caseins and whey proteins with no match within the human milk proteome were identified in the breast milk samples. These peptides were missing in the breast milk obtained from the mother after a prolonged milk- and dairy-free diet (three samples). The time course of cow's milk-derived β-Lg f(125–135) and β-casein f(81–92) in breast milk was determined from the MS ion intensity of the peptide signals. No intact cow's milk gene products were detected by HPLC-MS/MS analysis and Western blotting with anti-β-Lg antibody, but dot-blot analysis confirmed the occurrence of β-Lg fragments in the enriched peptide fraction of breast milk. These data suggest shifting the analytical perspective for the detection of dietary food allergens in breast milk from intact proteins to digested peptide fragments. The possible sensitization and elicitation potential or the tolerogenic properties of such low amounts of dietary peptides for the breastfed newborns remain to be explored.

Phosphatidylinositol 3-kinase-related kinases (PIKKs) play vital roles in the regulation of cell growth, proliferation, survival, and consequently metabolism, as well as in the cellular response to stresses such as ionizing radiation or redox changes. In humans six family members are known to date, namely mammalian/mechanistic target of rapamycin (mTOR), ataxia-telangiectasia mutated (ATM), ataxia- and Rad3-related (ATR), DNA-dependent protein kinase catalytic subunit (DNA-PKcs), suppressor of morphogenesis in genitalia-1 (SMG-1), and transformation/transcription domain-associated protein (TRRAP). All fulfill rather diverse functions and most of them have been detected in different cellular compartments including various cellular membranes. It has been suggested that the regulation of the localization of signaling proteins allows for generating a locally specific output. Moreover, spatial partitioning is expected to improve the reliability of biochemical signaling. Since these assumptions may also be true for the regulation of PIKK function, the current knowledge about the regulation of the localization of PIKKs at different cellular (membrane) compartments by a network of interactions is reviewed. Membrane targeting can involve direct lipid-/membrane interactions as well as interactions with membrane-anchored regulatory proteins, such as, for example, small GTPases, or a combination of both.

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Analyzing an in vitro gastroduodenal digest of whey proteins by high-performance liquid chromatography (HPLC) coupled to high-resolution/high-sensitivity tandem mass spectrometry (MS/MS), we sought to evaluate if state-of-art peptidomics provide comprehensive peptide coverage of food "digestomes". A multitude of small-sized peptides derived from both α-lactalbumin and β-lactoglobulin as well as disulfide cross-linked hetero-oligomers remained unassigned, even when the digests were compared before and after S−S reduction. The precipitation with 12% trichloroacetic acid demonstrated the occurrence of large-sized polypeptides that escaped the bioinformatic identification. The analysis of a HPLC−MS/MS run with different proteomic search engines generated dissimilar peptide subsets, thus emphasizing the demand of refined searching algorithms. Although the MS/MS fragmentation of monocharged ions with exclusion of non-peptide-interfering compounds enlarged the inventory of short peptides, the overall picture of the "digestome" was still incomplete. These findings raise relevant implications for the identification of possible food-derived bioactive peptides or allergenic determinants.

Increased efforts have been undertaken to better understand the formation of signaling complexes at cellular membranes. Since the preparation of proteins containing a transmembrane domain or a prenylation motif is generally challenging an alternative membrane anchoring unit that is easy to attach, water-soluble and binds to different membrane mimetics would find broad application. The 33-residue long FATC domain of yeast TOR1 (y1fatc) fulfills these criteria and binds to neutral and negatively charged micelles, bicelles, and liposomes. As a case study, we fused it to the FKBP506-binding region of the protein FKBP38 (FKBP38-BD) and used H- N NMR spectroscopy to characterize localization of the chimeric protein to micelles, bicelles, and liposomes. Based on these and published data for y1fatc, its use as a C-terminally attachable membrane anchor for other proteins is compatible with a wide range of buffer conditions (pH circa 6-8.5, NaCl 0 to >150 mM, presence of reducing agents, different salts such as MgCl and CaCl ). The high water-solubility of y1fatc enables its use for titration experiments against a membrane-localized interaction partner of the fused target protein. Results from studies with peptides corresponding to the C-terminal 17-11 residues of the 33-residue long domain by 1D H NMR and CD spectroscopy indicate that they still can interact with membrane mimetics. Thus, they may be used as membrane anchors if the full y1fatc sequence is disturbing or if a chemically synthesized y1fatc peptide shall be attached by native chemical ligation, for example, unlabeled peptide to N-labeled target protein for NMR studies.

No abstract

Most of the bread on the market contains several additional ingredients compared to those used for traditional baking (flour, water, yeast, and salt). Modern bread is often baked using enzymatic dough improvers, which, as technological aids, can be omitted on the label. Baking mixes also can contain varying percentages of hard fat or their derivatives. The possible animal origin of enzymes or fats in bread might remain unknown, raising ethical concerns for some categories of consumers. Herein, an array of analytical methodologies recently exploited to disclose the origin of enzymes in dough improvers has been extended to the detection of pig-derived ingredients in bread. PCR amplification of a mitochondrial cytochrome b (mt-Cytb) gene region enabled the detection of even trace amounts of porcine DNA in bread. Porcine pancreatic α-amylase was detected in bread spiked with porcine pancreatic enzymes using both Western blot and HPLC-tandem mass spectrometry-based targeted or untargeted proteomics. Attenuated total reflectance-Fourier transform infrared (ATR-FTIR) enabled quick discrimination between lardcontaining (1%, w/w) and conventional bread. However, gas chromatographic analysis of fatty acids produced characteristic patterns for bread baked with lard or other oils.

Ras homolog enriched in brain (Rheb) is a small GTPase that regulates mammalian/mechanistic target of rapamycin complex 1 (mTORC1) and, thereby, cell growth and metabolism. Here we show that cycling between the inactive GDP‐ and the active GTP‐bound state modulates the backbone dynamics of a C‐terminal truncated form, RhebΔCT, which is suggested to influence its interactions. We further investigated the interactions between RhebΔCT and the proposed Rheb‐binding domain of the regulatory protein FKBP38. The observed weak interactions with the GTP‐analogue‐ (GppNHp‐) but not the GDP‐bound state, appear to accelerate the GDP to GTP exchange, but only very weakly compared to a genuine GEF. Thus, FKBP38 is most likely not a GEF but a Rheb effector that may function in membrane targeting of Rheb.