Dereplikace látek a de novo charakterizace malých molekul z hmotnostních spekter

We describe the molecular dereplication principles and de novo characterization of small molecules obtained from liquid-chromatography mass spectrometry and imaging mass spectrometry data sets. Our methodology aims at supporting chemists and computer programmers to understand the hidden computing algorithms used for metabolomics mass spectrometry data processing. The approaches have been made available in the open-source tool CycloBranch. The presented tutorial extends the interpretation of mass spectra portfolia described in a series of papers published in Chemicke Listy, issues 2/2020 and 3/2020.

Metabolite Profiling and Classification of Highbush Blueberry Leaves under Different Shade Treatments

Blueberry belongs to the genus Vaccinium L. in the Ericaceae and is an economically important shrub that produces small berries that are rich in nutrients. There were differences in the appearance of blueberry leaves under different shade treatments. To explore the differences in metabolites in blueberry leaves under different shading treatments, nontargeted liquid chromatography–mass spectrometry (LC–MS) metabonomic analysis was performed. Different shade intensities resulted in significant differences in the contents of metabolites. A total of 6879 known metabolites were detected, including 750 significantly differentially expressed metabolites, including mainly lipids and lipid-like molecules and phenylpropanoid and polyketide superclass members. Based on a Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, the flavone and flavonol biosynthesis pathways were the most significantly enriched. The results of this study provide a reference and scientific basis for the establishment of a high-quality and high-yield shaded blueberry cultivation system.

Internal Standard an Important Analyte Use in Drug Analysis by Liquid Chromatography Mass Spectrometry- An Article

Internal standard is an external compound which is mixed with targeted analytical solution and matrix as a constant concentration and use for preparing calibration standard curve by using ratio of analyte area and internal standard area with analyte concentration and internal standard concentration. This calibration curve used for quantification of unknown concentration of anlayte of interest. This article provide necessary information about internal standard like its selection procedure, characterization, types and response factor , to all analyst who are connected with drug analysis. This article is more important and I think first article which focuses a clear idea about internal standard use in drug analysis.

Acetylation of lysines on affinity-purification matrices to reduce co-digestion of bead-bound ligands v1

In mass-spectrometry-based interaction proteomics on-bead digestion protocols are commonly applied after affinity-enrichment due to their simplicity and high efficiency. However, on-bead digestion often leads to strong background signals due to co-digestion of the bead-bound ligands such as streptavidin or antibodies. We present an effective, rapid and low-cost method to specifically reduce the peptide signals from co-digested matrix ligands. A short pre-incubation of matrix beads with Sulfo-NHS-Acetate (S-NHS-Ac) leads to acetylation of free amines on lysine side-chains of the bead-bound ligands making them resistant to Lys-C-mediated proteolysis. After binding of bait proteins to the acetylated beads we employ a two-step digestion protocol with the sequential use of Lys-C protease for on-bead digestion followed by in-solution digestion with trypsin. The strong reduction of interfering ligand peptides improves signal strength and data quality for the peptides of interest in liquid chromatography mass spectrometry (LC-MS).

Characterization of Monoclonal Antibody Glycan Heterogeneity Using Hydrophilic Interaction Liquid Chromatography-Mass Spectrometry

Glycosylation is a critical quality attribute of monoclonal antibody (mAb) therapeutics. Hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS) is an invaluable technology for the characterization of protein glycosylation. HILIC/MS-based glycan analysis relies on the library search using Glucose Units (GU) and accurate mass (AM) as the primary search parameters for identification. However, GU-based identifications are gradient-dependent and are not suitable for applications where separation gradients need to be optimized to analyze complex samples or achieve higher throughput. Additionally, the workflow requires calibration curves (using dextran ladder) to be generated for each analysis campaign, which in turn, are used to derive the GU values of the separated glycan species. To overcome this limitation, we employed a two-step strategy for targeted glycan analysis of a mAb expressed in Chinese Hamster Ovary (CHO) cells. The first step is to create a custom library of the glycans of interest independent of GU values (thereby eliminating the need for a calibration curve) and instead uses AM and retention time (RT) as the primary search variables. The second step is to perform targeted glycan screening using the custom-built library. The developed workflow was applied for targeted glycan analysis of a mAb expressed in CHO for 1) cell line selection 2) characterizing the day-wise glycan evolution in a model mAb during a fed-batch culture, 3) assessing the impact of different media conditions on glycosylation, and 4) evaluating the impact of two different process conditions on glycosylation changes in a model mAb grown in a bioreactor. Taken together, the data presented in this study provides insights into the sources of glycan heterogeneity in a model mAb that are seen during its commercial manufacturing.