Tim Causon

As part of established biomanufacturing development, screening and early phase bioprocess development occurs at bench scale (microplates and shake flasks) whereby conventional offline sampling can only provide limited feedback on fermentation bioprocess parameters including strain productivity. To address these limitations, a new sensitive and selective online analytical platform consisting entirely of commercially available components with a small footprint (valves, 2DLC hardware, LC separation, and online tandem mass spectrometry) was developed for online monitoring of chip-based microbioreactors. Fermentations of microbial cell factories (Saccharomyces cerevisiae) were cultivated in 20 μL bioreactors, requiring perfusion of cell culture media at low μL/min rates delivered by syringe pump modules, operated in a multiplexed configuration with a flow-through stream selection valve, and monitored with a 2DLC-MS/MS system adapted for microscale operation. This allows uninterrupted multiplexed microperfusions to be monitored with online measurements of metabolites from parallel fermentations without the occurrence of blockages or cross-contamination between independent fermentations. Fermentations of lactic-acid-producing strains ofS. cerevisiaewere continuously monitored over 5-24 h, demonstrating the suitability of the platform for online monitoring of product quantity and key metabolites for fermentation biotechnology. Offering minimal consumption of biological material and using <1.5 mL of cell culture media over 24 h per experiment, this new platform can be used for monitoring a broad range of biomolecules, rapid strain selection, and screening of microenvironmental factors and is adaptable for targeting other key biotechnology products.

A combination of ion mobility-mass spectrometry (IM-MS) measurements and computational methods were used to study structural and physicochemical properties of a range of quinoline-based drugs: amodiaquine (AQ), cinchonine (CIN), chloroquine (CQ), mefloquine (MQ), pamaquine (PQ), primaquine (PR), quinacrine (QR), quinine (QN), and sitamaquine (SQ). In experimental studies, ionization of these compounds using atmospheric pressure chemical ionization (APCI) yields monoprotonated species in the gas phase while electrospray ionization (ESI) also produces diprotonated forms of AQ, CQ, and QR and also for PQ, SQ, and QN in the presence of formic acid as an additive. Comparison of the trajectory-method-calculated and experimental IM-derived collisional cross sections (CCS_N2) were used to assign both the protonation sites and conformer geometry of all drugs considered with biases of 0.7-2.8% between calculated and experimental values. It was found that, in solution, AQ and QR are protonated at the ring nitrogen of the quinoline group, whereas the other drugs are protonated at the amine group of the alkyl chain. Finally, the conformers of [M + H]⁺ and [M + 2H]²⁺ assigned according to the lowest energies and CCS_N2 calculations were used to calculate the pK_a values of the antimalarial drugs and the relative abundance of these ions at different pH values that provided validation of the computational and experimental IM-MS results.

In addition to providing critical knowledge of the accurate mass of ions, ion mobility‐mass spectrometry (IM‐MS) delivers complementary data relating to the conformation and size of ions in the form of an ion mobility spectrum and derived parameters, namely, the ion's mobility (K) and the IM‐derived collision cross section (CCS). However, the maximum amount of information obtained in IM‐MS measurements is not currently transferred into analytical databases including the full mobility spectra (CCS distributions) as well as capturing of additional ion species (e.g., adducts) into the same compound entry. We introduce CCSfind, a new tool for building comprehensive databases from experimental IM‐MS measurements of small molecules. CCSfind allows predicted ion species to be chosen for input chemical formulae, which are then targeted by CCSfind after parsing open source mzML input files to provide a unified set of results within a single data processing step. CCSfind can handle both chromatographically separated isomers and IM separation of isomeric ions (e.g., “protomers” or conformers of the same ion species) with simple user control over the output for new database entries in SQL format. Files of up to 1 GB can be processed in less than 2 min on a desktop computer with 32 GB RAM with computational time scaling linearly with the size of the input mzML file or the number of input molecular formulae. Results are manually reviewed, annotated with experimental settings, before committing the database where the full dataset can be retrieved.

Ion mobility-mass spectrometry (IM-MS) offers benefits for lipidomics by obtaining IM-derived collision cross sections (CCS), a conditional property of an ion that can enhance lipid identification. While drift tube (DT) IM-MS retains a direct link to the primary experimental method to derive CCS values, other IM technologies rely solely on external CCS calibration, posing challenges due to dissimilar chemical properties between lipids and calibrants. To address this, we introduce MobiLipid, a novel tool facilitating the CCS quality control of IM-MS lipidomics workflows by internal standardization. MobiLipid utilizes a newly established ^DTCCS_N2 library for uniformly (U)¹³C-labeled lipids, derived from a U¹³C-labeled yeast extract, containing 377 ^DTCCS_N2 values. This automated open-source R Markdown tool enables internal monitoring and straightforward compensation for CCS_N2 biases. It supports lipid class- and adduct-specific CCS corrections, requiring only three U¹³C-labeled lipids per lipid class-adduct combination across 10 lipid classes without requiring additional external measurements. The applicability of MobiLipid is demonstrated for trapped IM (TIM)-MS measurements of an unlabeled yeast extract spiked with U¹³C-labeled lipids. Monitoring the CCS_N2 biases of ^TIMCCS_N2 values compared to ^DTCCS_N2 library entries utilizing MobiLipid resulted in mean absolute biases of 0.78% and 0.33% in positive and negative ionization mode, respectively. By applying the CCS correction integrated into the tool for the exemplary data set, the mean absolute CCS_N2 biases of 10 lipid classes could be reduced to approximately 0%.

Ion mobility-mass spectrometry (IM-MS) offers benefits for lipidomics by obtaining IM-derived collision cross sections (CCS), a conditional physicochemical parameter of an ion which can enhance lipid identification. While drift tube (DT) IM-MS retains a direct link to the primary experimental method to derive CCS values, other IM technologies rely solely on external CCS calibration, posing challenges due to dissimilar chemical properties between lipids and calibrants. To address this, we introduce MobiLipid, a novel tool facilitating CCS quality control of IM-MS lipidomics workflows by internal standardization. MobiLipid utilizes a newly established DTCCSN2 library for uniformly (U)13C labeled lipids, derived from a U13C labeled yeast extract, containing 377 DTCCSN2 values. This automated open-source R markdown tool enables internal monitoring and straightforward compensation for CCSN2 biases. It supports lipid class- and adduct-specific CCS corrections, requiring only three U13C labeled lipids per lipid class-adduct combination across 10 lipid classes, without requiring additional external measurements. The applicability of MobiLipid is demonstrated for trapped IM (TIM)-MS measurements of an unlabeled yeast extract spiked with U13C labeled lipids. Monitoring the CCSN2 biases of TIMCCSN2 values compared to DTCCSN2 library entries utilizing MobiLipid resulted in mean absolute biases of 0.78% and 0.33% in positive and negative ionization mode, respectively. By applying the CCS correction integrated into the tool for the exemplary dataset, the mean absolute CCSN2 biases of 10 lipid classes could be reduced to approximately 0%.

In addition to providing critical knowledge of the accurate mass of ions, ion mobility-mass spectrometry (IM-MS) delivers complementary data relating to the conformation and size of ions in the form of an ion mobility spectrum and derived parameters; namely the ion’s mobility (K) and the IM-derived collision cross section (CCS). However, the maximum amount of information obtained in IM-MS measurements is not currently transferred into analytical databases including the full mobility spectra (CCS distributions) as well as capturing of additional ion species (e.g., adducts) into the same compound entry. We introduce CCSfind, a new tool for building comprehensive databases from experimental IM-MS measurements of small molecules. CCSfind allows predicted ion species to be chosen for input chemical formulae, which are then targeted by CCSfind after parsing open source mzML input files to provide a unified set of results within a single data processing step. CCSfind can handle both chromatographically separated isomers and IM separation of isomeric ions (e.g., “protomers” or conformers of the same ion species) with simple user control over the output for new database entries in SQL format. Files of up to 1 GB can be processed in less than 2 minutes on a desktop computer with 32GB RAM with computational time scaling linearly with the size of the input mzML file or the number of input molecular formulae. Results are manually reviewed, annotated with experimental settings, before committing the database where the full dataset can be retrieved.

Fast liquid chromatography (LC) amino acid enantiomer separation of 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) derivatives using a chiral core-shell particle tandem column with weak anion exchange and zwitterionic-type quinine carbamate selectors in less than 3 min was achieved. Enantiomers of all AQC-derivatized proteinogenic amino acids and some isomeric ones (24 in total plus achiral glycine) were baseline separated (R_s > 1.5 except for glutamic acid with R_s = 1.3), while peaks of distinct amino acids and structural isomers (constitutional isomers and diastereomers of leucine and threonine) of the same configuration overlapped to various degrees. For this reason, drift tube ion mobility-mass spectrometry was added (i.e., LC-IM-MS) as an additional selectivity filter without extending run time. The IM separation dimension in combination with high-resolution demultiplexing enabled confirmation of threonine isomers (threonine, allo-threonine, homoserine), while leucine, isoleucine, and allo-isoleucine have almost identical collisional cross-section (^DTCCS_N2) values and added no selectivity to the partial LC separation. Density functional theory (DFT) calculations show that IM separation of threonine isomers was possible due to conformational stabilization by hydrogen bond formation between the hydroxyl side chain and the urea group. Generally, the CCS_N2 of protonated ions increased uniformly with addition of the AQC label, while outliers could be explained by consideration of intramolecular interactions and additional structural analysis. Preliminary validation of the enantioselective LC-IM-MS method for quantitative analysis showed compliance of accuracy and precision with common limits in bioanalytical methods, and applicability to a natural lipopeptide and a therapeutic synthetic peptide could be demonstrated.