Addressing Acid-Catalyzed Deamidation and the Solubility of Hydrophobic Peptides in Multi-Attribute Method Workflows.

Recently, we introduced an optimized and automated Multi-Attribute Method (MAM) workflow, which (a) significantly reduces the number of missed cleavages using an automated two-step digestion procedure and (b) dramatically reduces chromatographic peak tailing and carryover of hydrophobic peptides by implementing less retentive reversed-phase column chemistries. Here, further insights are provided on the impact of postdigest acidification and the importance of maintaining hydrophobic peptides in solution using strong chaotropic agents after digestion. We demonstrate how oxidation can significantly increase the solubility of hydrophobic peptides, a fact that can have a profound impact on quantitation of oxidation levels if care is not taken in MAM workflows.

Optimized Multi-Attribute Method Workflow Addressing Missed Cleavages and Chromatographic Tailing/Carry-Over of Hydrophobic Peptides.

Peptide mapping by liquid chromatography mass spectrometry (LC-MS) and the related multi-attribute method (MAM) are well-established analytical tools for verification of the primary structure and mapping/quantitation of co- and post-translational modifications (PTMs) or product quality attributes in biopharmaceutical development. Proteolytic digestion is a key step in peptide mapping workflows, which traditionally is labor-intensive, involving multiple manual steps. Recently, simple high-temperature workflows with automatic digestion were introduced, which facilitate robustness and reproducibility across laboratories.

Hybridization and complex evolution of Barbarea vulgaris and related species (Brassicaceae).

Barbarea, winter-cress, is a genus of 29 species in Brassicaceae, the mustard family, which has emerged as a model for evolution of plant defence and specialised metabolites. Notably, some Barbarea species have evolved the ability to produce triterpenoid saponins as the only ones in Brassicaceae, some of which make plants resistant to important herbivores. Resistance has, however, been lost in a distinct group of plants within B.

Characterization of Protein Glycoforms at Intact Level by Orbitrap Mass Spectrometry.

Intact mass analysis of proteins is simple, fast, and specific, and it effectively provides structural insight into the proteoforms or variants of the analyzed protein. For instance, the multiple glycoforms of recombinant monoclonal antibodies can be effectively analyzed by intact mass spectrometry (MS). A recent development in the Orbitrap technology has made this platform particularly well suited for analysis of large intact biomolecules, and here we describe procedures for performing intact mass analysis of intact glycoproteins using the Orbitrap platform, with the aim of identifying and quantitating the glycoforms.

Glucosinolate profiles and phylogeny in Barbarea compared to other tribe Cardamineae (Brassicaceae) and Reseda (Resedaceae), based on a library of ion trap HPLC-MS/MS data of reference desulfoglucosinolates.

A library of ion trap MS2 spectra and HPLC retention times reported here allowed distinction in plants of at least 70 known glucosinolates (GSLs) and some additional proposed GSLs. We determined GSL profiles of selected members of the tribe Cardamineae (Brassicaceae) as well as Reseda (Resedaceae) used as outgroup in evolutionary studies. We included several accessions of each species and a range of organs, and paid attention to minor peaks and GSLs not detected.

Comparison of glucosinolate diversity in the crucifer tribe Cardamineae and the remaining order Brassicales highlights repetitive evolutionary loss and gain of biosynthetic steps.

We review glucosinolate (GSL) diversity and analyze phylogeny in the crucifer tribe Cardamineae as well as selected species from Brassicaceae (tribe Brassiceae) and Resedaceae. Some GSLs occur widely, while there is a scattered distribution of many less common GSLs, tentatively sorted into three classes: ancient, intermediate and more recently evolved. The number of conclusively identified GSLs in the tribe (53 GSLs) constitute 60% of all GSLs known with certainty from any plant (89 GSLs) and apparently unique GSLs in the tribe constitute 10 of those GSLs conclusively identified (19%).

Molecular evidence of the hybrid origin of Cryptocoryne ×purpurea Ridl. nothovar. purpurea (Araceae).

Natural hybridization has been considered a source of taxonomic complexity in Cryptocoryne. A combined study of DNA sequencing data from the internal transcribed spacer (ITS) of nuclear ribosomal DNA and the trnK-matK region of chloroplast DNA was used to identify the parents of Cryptocoryne putative hybrids from Peninsular Malaysia. Based on the intermediate morphology and sympatric distribution area, the plants were tentatively identified as the hybrid Cryptocoryne ×purpurea nothovar.

Inter-laboratory study of an optimised peptide mapping workflow using automated trypsin digestion for monitoring monoclonal antibody product quality attributes.

Peptide mapping analysis is a regulatory expectation to verify the primary structure of a recombinant product sequence and to monitor post-translational modifications (PTMs). Although proteolytic digestion has been used for decades, it remains a labour-intensive procedure that can be challenging to accurately reproduce. Here, we describe a fast and reproducible protocol for protease digestion that is automated using immobilised trypsin on magnetic beads, which has been incorporated into an optimised peptide mapping workflow to show method transferability across laboratories.

Genome-Wide Association Studies in Apple Reveal Loci for Aroma Volatiles, Sugar Composition, and Harvest Date.

Understanding the genetic architecture of fruit quality traits is crucial to target breeding of apple ( L.) cultivars. We linked genotype and phenotype information by combining genotyping-by-sequencing (GBS) generated single nucleotide polymorphism (SNP) markers with fruit flavor volatile data, sugar and acid content, and historical trait data from a gene bank collection.

Population structure, relatedness and ploidy levels in an apple gene bank revealed through genotyping-by-sequencing.

In recent years, new genome-wide marker systems have provided highly informative alternatives to low density marker systems for evaluating plant populations. To date, most apple germplasm collections have been genotyped using low-density markers such as simple sequence repeats (SSRs), whereas only a few have been explored using high-density genome-wide marker information. We explored the genetic diversity of the Pometum gene bank collection (University of Copenhagen, Denmark) of 349 apple accessions using over 15,000 genome-wide single nucleotide polymorphisms (SNPs) and 15 SSR markers, in order to compare the strength of the two approaches for describing population structure.

Glucosinolate diversity within a phylogenetic framework of the tribe Cardamineae (Brassicaceae) unraveled with HPLC-MS/MS and NMR-based analytical distinction of 70 desulfoglucosinolates.

As a basis for future investigations of evolutionary trajectories and biosynthetic mechanisms underlying variations in glucosinolate structures, we screened members of the crucifer tribe Cardamineae by HPLC-MS/MS, isolated and identified glucosinolates by NMR, searched the literature for previous data for the tribe, and collected HPLC-MS/MS data for nearly all glucosinolates known from the tribe as well as some related structures (70 in total). This is a considerable proportion of the approximately 142 currently documented natural glucosinolates. Calibration with authentic references allowed distinction (or elucidation) of isomers in many cases, such as distinction of β-hydroxyls, methylthios, methylsulfinyls and methylsulfonyls.

A high-throughput method for genotyping S-RNase alleles in apple.

We present a new efficient screening tool for detection of S-alleles in apple. The protocol using general and multiplexed primers for PCR reaction and fragment detection on an automatized capillary DNA sequencer exposed a higher number of alleles than any previous studies. Analysis of alleles is made on basis of three individual fragment sizes making the allele interpretation highly accurate.

Specific glucosinolate analysis reveals variable levels of epimeric glucobarbarins, dietary precursors of 5-phenyloxazolidine-2-thiones, in watercress types with contrasting chromosome numbers.

Watercress obtained in food stores in the United States contained significant levels of epiglucobarbarin [(R)-2-hydroxy-2-phenylethylglucosinolate] and low levels of the 2S-epimer glucobarbarin identified by an HPLC+NMR+MS/MS approach. Typical combined levels were 4-7 μmol/g dry wt. The hydrolysis product, 5-phenyloxazolidine-2-thione (barbarin), was detected at similar levels as the precursor glucosinolates after autolysis of fresh watercress in water.

Expression of KxhKN4 and KxhKN5 genes in Kalanchoë blossfeldiana 'Molly' results in novel compact plant phenotypes: towards a cisgenesis alternative to growth retardants.

Many potted plants like Kalanchoë have an elongated natural growth habit, which has to be controlled through the application of growth regulators. These chemicals will be banned in the near future in all the EU countries. Besides their structural functions, the importance of homeotic genes to modify plant architecture appears evident.

Genetic and sexual separation between insect resistant and susceptible Barbarea vulgaris plants in Denmark.

Co-evolution between herbivores and plants is believed to be one of the processes creating Earth's biodiversity. However, it is difficult to disentangle to what extent diversification is really driven by herbivores or by other historical-geographical processes like allopatric isolation. In the cruciferous plant Barbarea vulgaris, some Danish individuals are resistant to herbivory by flea beetles (Phyllotreta nemorum), whereas others are not.

Variable glucosinolate profiles of Cardamine pratensis (Brassicaceae) with equal chromosome numbers.

A novel glucosinolate, 3-(hydroxymethyl)pentylglucosinolate, was present at high levels in Cardamine pratensis L. from eastern North America and in commercially obtained seeds, but not in C. pratensis plants from southern Scandinavia.

Meiotic analysis of Danish species of Barbarea (Brassicaceae) using FISH: chromosome numbers and rDNA sites.

PMCs of 5 taxa of Barbarea from Denmark, i.e. B.

Cytogenetics of Danish species of Barbarea (Brassicaceae): chromocentres, chromosomes and rDNA sites.

Seventeen Danish accessions of five taxa of Barbarea, i.e. B.

The genomic organization and evolutionary distribution of a tandemly repeated DNA sequence family in the genus Crocus (Iridaceae).

From a recombinant DNA-library from Crocus vernus, two closely related clones of highly repetitive DNA, pCvKB7 and pCvKB8, were sequenced and their genomic distribution and organization were investigated by Southern and in situ hybridization. The lengths of the clones were 181 and 178 bp respectively; the sequences were approximately 85% identical, and thus belonged to a sequence family, named the pCvKB8-family. No homologous sequences were found in the databases (BLAST made may 2004).

Glucosinolates, flea beetle resistance, and leaf pubescence as taxonomic characters in the genus Barbarea (Brassicaceae).

Glucosinolate content of leaves and roots, diversity in leaf pubescence, and resistance to two near-isogenic lines of the flea beetle Phyllotreta nemorum with or without an R-gene, were determined for 27 accessions of 7 Barbarea taxa, i.e. B.

Genome discrimination by in situ hybridization in Icelandic species of Elymus and Elytrigia (Poaceae: Triticeae).

The genome constitution of Icelandic Elymus caninus, E. alaskanus, and Elytrigia repens was examined by fluorescence in situ hybridization using genomic DNA and selected cloned sequences as probes. Genomic in situ hybridization (GISH) of Hordeum brachyantherum ssp.

Unifying plant molecular data and plants.

Located at a botanical department at an Agricultural University, our taxonomical and genetic research is mainly directed towards cultivated plants and their wild relatives. The investigations are usually under a common heading 'experimental taxonomy', and include basic systematics, cytogenetics, biodiversity, population dynamics, conservation and evolutionary questions correlating the wild species and the cultivated forms. Our point of initiation is the plants and questions/problems raised regarding these plants.