Today’s highly accurate spectra provided by modern tandem mass spectrometers offer considerable advantages for the analysis of proteomic samples of increased complexity. Among other factors, the quantity of reliably identified peptides is considerably influenced by the peptide identification algorithm. While most widely used search engines were developed when high-resolution mass spectrometry data were not readily available for fragment ion masses, we have designed a scoring algorithm particularly suitable for high mass accuracy. Our algorithm, MS Amanda, is generally applicable to HCD, ETD, and CID fragmentation type data. The algorithm confidently explains more spectra at the same false discovery rate than Mascot or SEQUEST on examined high mass accuracy data sets, with excellent overlap and identical peptide sequence identification for most spectra also explained by Mascot or SEQUEST. MS Amanda, available at http://ms.imp.ac.at/?goto=msamanda, is provided free of charge both as standalone version for integration into custom workflows and as a plugin for the Proteome Discoverer platform.

Summary: A common argument against using plants as a production system for therapeutic proteins is their inability to perform authentic human N‐glycosylation (i.e. the presence of β1,2‐xylosylation and core α1,3‐fucosylation). In this study, RNA interference (RNAi) technology was used to obtain a targeted down‐regulation of the endogenous β1,2‐xylosyltransferase (XylT) and α1,3‐fucosyltransferase (FucT) genes in Nicotiana benthamiana, a tobacco‐related plant species widely used for recombinant protein expression. Three glyco‐engineered lines with significantly reduced xylosylated and/or core α1,3‐fucosylated glycan structures were generated. The human anti HIV monoclonal antibody 2G12 was transiently expressed in these glycosylation mutants as well as in wild‐type plants. Four glycoforms of 2G12 differing in the presence/absence of xylose and core α1,3‐fucose residues in their N‐glycans were produced. Notably, 2G12 produced in XylT/FucT‐RNAi plants was found to contain an almost homogeneous N‐glycan species without detectable xylose and α1,3‐fucose residues. Plant‐derived glycoforms were indistinguishable from Chinese hamster ovary (CHO)‐derived 2G12 with respect to electrophoretic properties, and exhibited functional properties (i.e. antigen binding and HIV neutralization activity) at least equivalent to those of the CHO counterpart. The generated RNAi lines were stable, viable and did not show any obvious phenotype, thus providing a robust tool for the production of therapeutically relevant glycoproteins in plants with a humanized N‐glycan structure.

Pichia pastoris is widely used as a production platform for heterologous proteins and model organism for organelle proliferation. Without a published genome sequence available, strain and process development relied mainly on analogies to other, well studied yeasts like Saccharomyces cerevisiae.

Many therapeutic proteins are glycosylated and require terminal sialylation to attain full biological activity. Current manufacturing methods based on mammalian cell culture allow only limited control of this important posttranslational modification, which may lead to the generation of products with low efficacy. Here we report in vivo protein sialylation in plants, which have been shown to be well suited for the efficient generation of complex mammalian glycoproteins. This was achieved by the introduction of an entire mammalian biosynthetic pathway in Nicotiana benthamiana, comprising the coordinated expression of the genes for (i) biosynthesis, (ii) activation, (iii) transport, and (iv) transfer of Neu5Ac to terminal galactose. We show the transient overexpression and functional integrity of six mammalian proteins that act at various stages of the biosynthetic pathway and demonstrate their correct subcellular localization. Co-expression of these genes with a therapeutic glycoprotein, a human monoclonal antibody, resulted in quantitative sialylation of the Fc domain. Sialylation was at great uniformity when glycosylation mutants that lack plant-specific N-glycan residues were used as expression hosts. Finally, we demonstrate efficient neutralization activity of the sialylated monoclonal antibody, indicating full functional integrity of the reporter protein. We report for the first time the incorporation of the entire biosynthetic pathway for protein sialylation in a multicellular organism naturally lacking sialylated glycoconjugates. Besides the biotechnological impact of the achievement, this work may serve as a general model for the manipulation of complex traits into plants.