Filling the gaps in peptide maps with a platform assay for top-down characterization of purified protein samples.

Liquid chromatography-mass spectrometry (LC-MS) intact mass analysis and LC-MS/MS peptide mapping are decisional assays for developing biological drugs and other commercial protein products. Certain PTM types, such as truncation and oxidation, increase the difficulty of precise proteoform characterization owing to inherent limitations in peptide and intact protein analyses. Top-down MS (TDMS) can resolve this ambiguity via fragmentation of specific proteoforms. We leveraged the strengths of flow-programmed (fp) denaturing online buffer exchange (dOBE) chromatography, including robust automation, relatively high ESI sensitivity, and long MS/MS window time, to support a TDMS platform for industrial protein characterization. We tested data-dependent (DDA) and targeted strategies using 14 different MS/MS scan types featuring combinations of collisional- and electron-based fragmentation as well as proton transfer charge reduction. This large, focused dataset was processed using a new software platform, named TDAcquireX, that improves proteoform characterization through TDMS data aggregation. A DDA-based workflow provided objective identification of αLac truncation proteoforms with a two-termini clipping search. A targeted TDMS workflow facilitated the characterization of αLac oxidation positional isomers. This strategy relied on using sliding window-based fragment ion deconvolution to generate composite proteoform spectral match (cPrSM) results amenable to fragment noise filtering, which is a fundamental enhancement relevant to TDMS applications generally.

An extensive disulfide bond network prevents tail contraction in Agrobacterium tumefaciens phage Milano.

A contractile sheath and rigid tube assembly is a widespread apparatus used by bacteriophages, tailocins, and the bacterial type VI secretion system to penetrate cell membranes. In this mechanism, contraction of an external sheath powers the motion of an inner tube through the membrane. The structure, energetics, and mechanism of the machinery imply rigidity and straightness. The contractile tail of Agrobacterium tumefaciens bacteriophage Milano is flexible and bent to varying degrees, which sets it apart from other contractile tail-like systems. Here, we report structures of the Milano tail including the sheath-tube complex, baseplate, and putative receptor-binding proteins. The flexible-to-rigid transformation of the Milano tail upon contraction can be explained by unique electrostatic properties of the tail tube and sheath. All components of the Milano tail, including sheath subunits, are crosslinked by disulfides, some of which must be reduced for contraction to occur. The putative receptor-binding complex of Milano contains a tailspike, a tail fiber, and at least two small proteins that form a garland around the distal ends of the tailspikes and tail fibers. Despite being flagellotropic, Milano lacks thread-like tail filaments that can wrap around the flagellum, and is thus likely to employ a different binding mechanism.

Determination of Allergen Levels, Isoforms, and Their Hydroxyproline Modifications Among Peanut Genotypes by Mass Spectrometry.

The recently published reference genome of peanuts enables a detailed molecular description of the allergenic proteins of the seed. We used LC-MS/MS to investigate peanuts of different genotypes to assess variability and to better describe naturally occurring allergens and isoforms. Using relative quantification by mass spectrometry, minor variation of some allergenic proteins was observed, but total levels of Ara h 1, 2, 3, and 6 were relatively consistent among 20 genotypes. Previously published RP-HPLC methodology was used for comparison. The abundance of three Ara h 3 isoforms were variable among the genotypes and contributed to a large proportion of total Ara h 3 where present. Previously unpublished hydroxyproline sites were identified in Ara h 1 and 3. Hydroxylation did not vary significantly where sites were present. Peanut allergen composition was largely stable, with only some isoforms displaying differences between genotypes. The resulting differences in allergenicity are of unknown clinical significance but are likely to be minor. The data presented herein allow for the design of targeted MS methodology to allow the quantitation and therefore control of peanut allergens of clinical relevance and observed variability.

Shellfish Tropomyosin IgE Cross-Reactivity Differs Among Edible Insect Species.

SCOPE: Insects are a potentially environmentally friendly alternative dietary protein source to supplement mammalian and fish sources, but potential allergenic risks are a concern. Consumption of insects may result in anaphylaxis and has been implicated in cross-reactivity with shellfish. Many allergenic proteins may be involved in cross-reactivity, including tropomyosin (TM). The uniformity of TM cross-reactivity among edible insects is unknown. Candidate edible insects for variability in shellfish IgE cross-reactivity are investigated. METHODS AND RESULTS: Selected insects and known related sources of allergens are extracted and probed by immunoblot with sera/plasma from patients sensitized to insects or shellfish. Quantification of TM in these extracts is performed using mass spectrometry. A comparison of the quantity of TM and the IgE reactivity of TM from these insects is performed. Distinct patterns of IgE cross-reactivity are observed with three insect species showing diminished reactivity. This pattern is not consistent with the amount of TM present in these insects, or with overall sequence homology. CONCLUSION: Insects display a diversity of TM-associated IgE reactivity. It is likely that minor sequence features and/or structural effects are primarily responsible. Additionally, it is demonstrated that some insect species may present significantly less IgE cross-reactivity to shrimp than do others.