Gerrit Hermann

Maleimide-functionalised platinum(IV) complexes as a synthetic platform for targeted drug delivery

Maleimide-functionalised Pt(IV) complexes with highly selective binding properties to thiol groups were synthesised as precursors for binding of thiol-containing tumour-targeting molecules like human serum albumin.

METLIN: A Technology Platform for Identifying Knowns and Unknowns

METLIN originated as a database to characterize known metabolites and has since expanded into a technology platform for the identification of known and unknown metabolites and other chemical entities. Through this effort it has become a comprehensive resource containing over 1 million molecules including lipids, amino acids, carbohydrates, toxins, small peptides, and natural products, among other classes. METLINs high-resolution tandem mass spectrometry (MS/MS) database, which plays a key role in the identification process, has data generated from both reference standards and their labeled stable isotope analogues, facilitated by METLIN-guided analysis of isotope-labeled microorganisms. The MS/MS data, coupled with the fragment similarity search function, expand the tools capabilities into the identification of unknowns. Fragment similarity search is performed independent of the precursor mass, relying solely on the fragment ions to identify similar structures within the database. Stable isotope data also facilitate characterization by coupling the similarity search output with the isotopic m/ z shifts. Examples of both are demonstrated here with the characterization of four previously unknown metabolites. METLIN also now features in silico MS/MS data, which has been made possible through the creation of algorithms trained on METLINs MS/MS data from both standards and their isotope analogues. With these informatic and experimental data features, METLIN is being designed to address the characterization of known and unknown molecules.

Characterization of metal-tagged antibodies used in ICP-MS-based immunoassays

AbstractA detailed characterization of metal-tagged antibodies is the prerequisite for the implementation of quantitative concepts in inductively coupled plasma–mass spectrometry (ICP-MS)-based bioanalysis or future medical diagnosis. In this paper, the common modification with bifunctional ligands containing maleimide residues as a reactive group was investigated in detail via size exclusion chromatography (SEC)-ICP-MS and liquid chromatography–time-of-flight (LC-TOF)-MS to determine the preservation of the antibody structure after tagging. Mouse monoclonal IgG modified with metal-coded tags (MeCATs) was used as a model system. Several antibody fragments were identified carrying different numbers of metal tags. In a second step, a functionality test was performed with isolated fragments where the antigen specificity was tested in a dot blot immunoassay. FigureSEC-ICP-MS chromatogram of metal tagged antibody

In vivo synthesized 34S enriched amino acid standards for species specific isotope dilution of proteins

A generic quantification approach was introduced addressing the characterization of protein standards while fulfilling the principles of metrology. Traceable absolute quantification was achieved combining a proven biochemical method, i.e. protein hydrolysis followed by amino acid quantification with the concept of species specific isotope dilution analysis (IDA). The method relies on the determination of the two sulfur containing amino acids, cysteine and methionine by sulfur speciation analysis and is hence applicable to any protein containing sulfur. In vivo synthesis using 34S as sulfur source in yeast fermentations provided species specific isotopically enriched standards for IDA quantification of cysteine and methionine in the oxidized forms, methionine sulfone and cysteic acid. Reverse isotope dilution mass spectrometry (IDMS) characterization by inductively coupled plasma mass spectrometry (ICP-MS) combined to anion exchange showed that very high concentrated spike material could be produced with μmol amounts of proteinogenic sulfur containing amino acids per g cell dry weight. An enrichment of 34S to 96.3 ± 0.4% (n = 3) and 98.5 ± 0.4% (n = 3) for cysteic acid and methionine sulfone, respectively, was assessed. The established IDA method was validated for the absolute quantification of commercially available lysozyme and ceruloplasmin standards including the calculation of a total combined uncertainty budget.

A Novel Lipidomics Workflow for Improved Human Plasma Identification and Quantification Using RPLC-MSn Methods and Isotope Dilution Strategies

Lipid identification and quantification are essential objectives in comprehensive lipidomics studies challenged by the high number of lipids, their chemical diversity, and their dynamic range. In this work, we developed a tailored method for profiling and quantification combining (1) isotope dilution, (2) enhanced isomer separation by C30 fused-core reversed-phase material, and (3) parallel Orbitrap and ion trap detection by the Orbitrap Fusion Lumos Tribid mass spectrometer. The combination of parallelizable ion analysis without time loss together with different fragmentation techniques (HCD/CID) and an inclusion list led to higher quality in lipid identifications exemplified in human plasma and yeast samples. Moreover, we used lipidome isotope-labeling of yeast (LILY)-a fast and efficient in vivo labeling strategy in Pichia pastoris-to produce (nonradioactive) isotopically labeled eukaryotic lipid standards in yeast. We integrated the 13C lipids in the LC-MS workflow to enable relative and absolute compound-specific quantification in yeast and human plasma samples by isotope dilution. Label-free and compound-specific quantification was validated by comparison against a recent international interlaboratory study on human plasma SRM 1950. In this way, we were able to prove that LILY enabled quantification leads to accurate results, even in complex matrices. Excellent analytical figures of merit with enhanced trueness, precision and linearity over 4-5 orders of magnitude were observed applying compound-specific quantification with 13C-labeled lipids. We strongly believe that lipidomics studies will benefit from incorporating isotope dilution and LC-MSn strategies.

The study of reduced versus oxidized glutathione in cancer cell models employing isotopically labelled standards

In this work, LC-MS/MS assays for accurate quantification of underivatized glutathione (GSH) and its oxidized form glutathione disulfide (GSSG) were developed based on isotope dilution. Both hydrophilic interaction (HILIC) and reversed phase chromatography (RPC) were implemented. Different protocols dedicated to cancer cell lysis were validated in terms of extraction efficiency, recovery, and unwanted glutathione oxidation. The latter was monitored using isotopologues of GSSG, which were formed upon reaction with isotopically enriched GSH and natural GSH of the sample. Finally, LC-MS/MS was employed for studying the GSH : GSSG ratios in several cancer cells (HCT116, GLC4, and SW480) upon exposure to anticancer metallodrugs. Clinically well-established cis-diamine-dichloro-platinum(II) (cisplatin) and sodium trans-[tetrachloridobis(1H-indazole)ruthenate(III)] (KP1339), promising experimental drugs, were addressed. In both cases, a decrease of the GSH : GSSG ratio was observed upon drug exposure. It was more pronounced for cisplatin, where the ratio shifted from 440 : 1 to 240 : 1 and from 160 : 1 to 90 : 1 in HCT116 and GLC4 cells, respectively. For KP1339, a significant decrease was observed in the SW480 cancer cell model, whereas the change was not significant in HCT116 cells. Taken together, this study introduces a new sensitive and robust method for the evaluation of drug-induced changes in the intracellular GSH : GSSG ratio of human cells.

13 C-labelled yeast as internal standard for LC–MS/MS and LC high resolution MS based amino acid quantification in human plasma

HIGHLIGHTSQuantitative methods based on isotope dilution by HRMS and QQQ‐MS were developed for the analysis of underivatised amino acids in human plasma.A fully 13C labelled yeast extract provided internal standards for human plasma samples.For method validation a certified reference material for metabolites in human plasma was used.Reversed phase and hydrophilic interaction liquid chromatography were validated. ABSTRACT Extracts from isotopically labelled organisms can be a versatile source for isotopically labelled chemical compounds providing ideal internal standards in mass spectrometry based assays. In this work, the application of 13C enriched yeast (Pichia pastoris) for accurate absolute metabolite quantification in human samples was investigated. >99% 13C enriched Pichia pastoris was produced via fermentation and extracted employing established protocols. Quantitative assays based on LC‐triple quadrupole mass spectrometry (QQQ‐MS) and LC‐high resolution mass spectrometry (HRMS) were validated using the Standard Reference Material, SRM 1950 – metabolites in frozen human plasma. 14 amino acids (as given in the certificate) were quantified using separations by reversed phase liquid chromatography (RPLC) and hydrophilic interaction liquid chromatography (HILIC). The latter chromatographic separation provided retention and selectivity for the amino acid panel, while the studied approaches employing RPLC relied on the selectivity of the MS detection. Cross‐validation using the different MS platforms showed that in all cases the application of in‐vivo labelled standards resulted in a significant improvement of trueness and precision. LODs and LOQs ranged, regardless of the detection system and addition of internal standards, in the same order of magnitude. The linear dynamic range of the employed detection systems was enhanced at least for one order of magnitude for several analytes when the internal standards were applied.