A MALDI-MS Methodology for Studying Metabolic Heterogeneity of Single Cells in a Population.

Mass spectrometry based metabolomics is the highly multiplexed, label-free analysis of small molecules such as metabolites or lipids in biological systems, and thus one of the most direct ways to characterize phenotypes. However, the phenotyping of populations with single-cell resolution is a great challenge due to the small number of molecules contained in an individual cell. Here we describe a microarray-based sample preparation workflow for MALDI mass spectrometry that has single-cell sensitivity and allows high-throughput analysis of lipids and pigments in single algae cells. The microarray targets receive individual cells in 1430 separate spots that allow the cells to be lysed individually without cross-contamination. Using positive ion mode and 2,5-dihydroxybenzoic acid as the MALDI matrix, the mass spectra unveil information about the relative composition of more than 20 different lipids/pigments in each individual cell within the population. Thus, the method allows the analysis of cellular phenotypes in a population on a completely new level.

Monitoring of antibody glycosylation pattern based on microarray MALDI-TOF mass spectrometry.

Biologically manufactured monoclonal antibodies (mAb) can strongly vary in their efficacy and affinity. Therefore, engineering and production of the mAb is highly regulated and requires product monitoring, especially in terms of N-glycosylation patterns. In this work, we present a high-throughput matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) method based on a microarray technology to monitor N-glycopeptides of IgG1 produced in a perfusion cell culture. A bottom-up approach combined with zwitterionic-hydrophilic interaction liquid chromatography for sample purification was used to determine the day-by-day variation of the terminal galactose within two major N-glycoforms. Our results show that microarrays for mass spectrometry (MAMS) are a robust platform for the rapid determination of the carbohydrate distribution. The spectral repeatability is characterized by a low coefficient of variations (1.7% and 7.1% for the FA2 and FA2G1 structures, respectively) and allows to detect the N-glycosylation variability resulting from operating conditions during the bioreactor process. The observed trend of released N-glycans was confirmed using capillary gel electrophoresis with laser-induced fluorescence detection. Therefore, the microarray technology is a promising analytical tool for glycosylation control during the production process of recombinant proteins.

High-Throughput Monitoring of Cocaine and Its Metabolites in Hair Using Microarrays for Mass Spectrometry and Matrix-Assisted Laser Desorption/Ionization-Tandem Mass Spectrometry.

Because of inhomogeneous matrix-assisted laser desorption/ionization (MALDI) matrix crystallization and laser shot-to-shot variability, quantitation is not generally performed by MALDI mass spectrometry. Here we introduce a high-throughput MALDI method using an innovative high-density microarray for mass spectrometry (MAMS) technology, which allows semiquantitative measurement of cocaine and its metabolites, benzoylecgonine, cocaethylene, and ecgonine methyl ester. A MAMS slide containing lanes of hydrophilic spots and an automated slider to drag a sample droplet over several small spots can accomplish automatic sample aliquoting and lead to homogeneous crystallization of the matrix-analyte mixture and, thus, to a reproducible signal (average RSD 6%). Four hair samples of self-reported drug users were analyzed in parallel by MALDI-MS/MS and by a validated LC-MS/MS method. The consumption profiles as well as the metabolite-parent drug ratios obtained correlated well, confirming the effectiveness of the MALDI-MS/MS method to establish a calendar of consumption in only 1 mg of hair. The analysis time for 10 hair samples is below 40 min, with 12 replicates per sample. Since only 3 µL of a 20 µL extract is analyzed, complementary assays are possible, such as the detection of additional drugs. The semiquantitative MALDI method worked well with only a small amount of hair and gave results in less than 4 min per sample, including replicates. This was made possible by the use of MAMS slides for sample preparation, which thus present significant advantages over traditional methods in cases where results are required urgently or if samples are scarce.

Isotope labeling to determine the dynamics of metabolic response in CHO cell perfusion bioreactors using MALDI-TOF-MS.

The steady-state operation of Chinese hamster ovary (CHO) cells in perfusion bioreactors requires the equilibration of reactor dynamics and cell metabolism. Accordingly, in this work we investigate the transient cellular response to changes in its environment and their interactions with the bioreactor hydrodynamics. This is done in a benchtop perfusion bioreactor using MALDI-TOF MS through isotope labeling of complex intracellular nucleotides (ATP, UTP) and nucleotide sugars (UDP-Hex, UDP-HexNAc). By switching to a 13 C6 glucose containing feed media during constant operation at 20 × 106 cells and a perfusion rate of 1 reactor volume per day, isotopic steady state was studied. A step change to the 13 C6 glucose medium in spin tubes allowed the determination of characteristic times for the intracellular turnover of unlabeled metabolites pools, τST (≤0.56 days), which were confirmed in the bioreactor. On the other hand, it is shown that the reactor residence time τR (1 day) and characteristic time for glucose uptake τGlc (0.33 days), representative of the bioreactor dynamics, delayed the consumption of 13 C6 glucose in the bioreactor and thus the intracellular 13 C enrichment. The proposed experimental approach allowed the decoupling of bioreactor hydrodynamics and intrinsic dynamics of cell metabolism in response to a change in the cell culture environment. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1630-1639, 2017.

Single-Cell Mass Spectrometry of Metabolites Extracted from Live Cells by Fluidic Force Microscopy.

Single-cell metabolite analysis provides valuable information on cellular function and response to external stimuli. While recent advances in mass spectrometry reached the sensitivity required to investigate metabolites in single cells, current methods commonly isolate and sacrifice cells, inflicting a perturbed state and preventing complementary analyses. Here, we propose a two-step approach that combines nondestructive and quantitative withdrawal of intracellular fluid with subpicoliter resolution using fluidic force microscopy, followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The developed method enabled the detection and identification of 20 metabolites recovered from the cytoplasm of individual HeLa cells. The approach was further validated in 13C-glucose feeding experiments, which showed incorporation of labeled carbon atoms into different metabolites. Metabolite sampling, followed by mass spectrometry measurements, enabled the preservation of the physiological context and the viability of the analyzed cell, providing opportunities for complementary analyses of the cell before, during, and after metabolite analysis.

Intracellular CHO Cell Metabolite Profiling Reveals Steady-State Dependent Metabolic Fingerprints in Perfusion Culture.

Perfusion cell culture processes allow the steady-state culture of mammalian cells at high viable cell density, which is beneficial for overall product yields and homogeneity of product quality in the manufacturing of therapeutic proteins. In this study, the extent of metabolic steady state and the change of the metabolite profile between different steady states of an industrial Chinese hamster ovary (CHO) cell line producing a monoclonal antibody (mAb) was investigated in stirred tank perfusion bioreactors. Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) of daily cell extracts revealed more than a hundred peaks, among which 76 metabolites were identified by tandem MS (MS/MS) and high resolution Fourier transform ion cyclotron resonance (FT-ICR) MS. Nucleotide ratios (Uridine (U)-ratio, nucleotide triphosphate (NTP)-ratio and energy charge (EC)) and multivariate analysis of all features indicated a consistent metabolite profile for a stable culture performed at 40 × 106 cells/mL over 26 days of culture. Conversely, the reactor was operated continuously so as to reach three distinct steady states one after the other at 20, 60, and 40 × 106 cells/mL. In each case, a stable metabolite profile was achieved after an initial transient phase of approximately three days at constant cell density when varying between these set points. Clear clustering according to cell density was observed by principal component analysis, indicating steady-state dependent metabolite profiles. In particular, varying levels of nucleotides, nucleotide sugar, and lipid precursors explained most of the variance between the different cell density set points. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:879-890, 2017.

High-throughput profiling of nucleotides and nucleotide sugars to evaluate their impact on antibody N-glycosylation.

Recent advances in miniaturized cell culture systems have facilitated the screening of media additives on productivity and protein quality attributes of mammalian cell cultures. However, intracellular components are not routinely measured due to the limited throughput of available analytical techniques. In this work, time profiling of intracellular nucleotides and nucleotide sugars of CHO-S cell fed-batch processes in a micro-scale bioreactor system was carried out using a recently developed high-throughput method based on matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOF-MS). Supplementation of various media additives significantly altered the intracellular nucleotides and nucleotide sugars that are inextricably linked to the process of glycosylation. The results revealed that UDP-Gal synthesis appeared to be particularly limiting whereas the impact of elevated UDP-GlcNAc and GDP-Fuc levels on the final glycosylation patterns was only marginally important. In contrast, manganese and asparagine supplementation altered the glycan profiles without affecting intracellular components. The combination of miniaturized cell cultures and high-throughput analytical techniques serves therefore as a useful tool for future quality driven media optimization studies.

Single-cell MALDI Tandem Mass Spectrometry: Unambiguous Assignment of Small Biomolecules from Single Chlamydomonas reinhardtii Cells.

The analysis of compounds from single cells is a major challenge in analytical life science. Labeling strategies, for instance fluorescence detection, are well established for measuring proteins with single cell sensitivity, but they mostly fail to detect small molecules. More recently mass spectrometry has entered the realm of single cell sensitivity and enables the label-free and highly parallelized detection of small biomolecules from single cells. The assignment of signals detected in single cells, however, generally has to rely on measurements in whole cell culture extracts. Isobaric structures, contaminations, higher noise levels and the high variability in the abundance of peaks between single cells complicate the assignment of peaks in single-cell spectra. Tandem mass spectrometry would be very useful for compound identification via mass spectrometry directly in single-cell analyses. Here we present the first single cell tandem mass spectra collected using matrix-assisted laser-desorption/ionization. The spectra obtained allow the assignment of most compounds detected in the spectra. We also show that the fragmentation is not restricted to the most abundant peaks in the spectra, but over a dynamic range of more than one order of magnitude.

Differential Isotope Labeling of Glycopeptides for Accurate Determination of Differences in Site-Specific Glycosylation.

We introduce a stable isotope labeling approach for glycopeptides that allows a specific glycosylation site in a protein to be quantitatively evaluated using mass spectrometry. Succinic anhydride is used to specifically label primary amino groups of the peptide portion of the glycopeptides. The heavy form (D4(13)C4) provides an 8 Da mass increment over the light natural form (H4(12)C4), allowing simultaneous analysis and direct comparison of two glycopeptide profiles in a single MS scan. We have optimized a protocol for an in-solution trypsin digestion, a one-pot labeling procedure, and a post-labeling solid-phase extraction to obtain purified and labeled glycopeptides. We provide the first demonstration of this approach by comparing IgG1 Fc glycopeptides from polyclonal IgG samples with respect to their galactosylation and sialylation patterns using MALDI MS and LC-ESI-MS.

Microarray-based MALDI-TOF mass spectrometry enables monitoring of monoclonal antibody production in batch and perfusion cell cultures.

Cell culture process monitoring in monoclonal antibody (mAb) production is essential for efficient process development and process optimization. Currently employed online, at line and offline methods for monitoring productivity as well as process reproducibility have their individual strengths and limitations. Here, we describe a matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS)-based on a microarray for mass spectrometry (MAMS) technology to rapidly monitor a broad panel of analytes, including metabolites and proteins directly from the unpurified cell supernatant or from host cell culture lysates. The antibody titer is determined from the intact antibody mass spectra signal intensity relative to an internal protein standard spiked into the supernatant. The method allows a semi-quantitative determination of light and heavy chains. Intracellular mass profiles for metabolites and proteins can be used to track cellular growth and cell productivity.

Resolution pattern for mass spectrometry imaging.

RATIONALE: Up to now, there is no 'gold standard' for determining the resolution of a mass spectrometry imaging (MSI) setup (comprising the instrument, the sample preparation, the sample and the instrument settings). A standard sample in combination with a standard protocol to define the MSI resolution would be desirable in order to compare the setups of different laboratories, and as a regular quality control/performance check. METHODS: Microstructured resolution patterns were fabricated that can be used to determine the spatial resolution in MSI experiments, down to the range of a few µm. Two different strategies were employed, one where the resolution pattern is laser machined into a thin metal foil, which can be placed over a sample to be imaged, and a second one where hydrophilic grooves are machined into an omniphobic coating covering the surface of an indium tin oxide covered glass slide. When dragging a sample solution over the slide's surface, the sample is automatically retained in the hydrophilic grooves, but repelled by the omniphobic coating. RESULTS: The technology was tested on a commercial matrix-assisted laser desorption/ionization (MALDI) imaging instrument, and a spatial resolution in the vicinity of 50 µm was determined. The finest features of the microstructured resolution patterns are compatible with the best spatial resolution of MALDI imaging systems available to date. CONCLUSIONS: The use of metal resolution grids or glass slides with hydrophilic/hydrophobic structures is suitable for the convenient determination of the resolution limit of the MALDI imaging instrument as determined by its hardware. These structures are straightforward both to produce and to use.

Screening of Chlamydomonas reinhardtii Populations with Single-Cell Resolution by Using a High-Throughput Microscale Sample Preparation for Matrix-Assisted Laser Desorption Ionization Mass Spectrometry.

The consequences of cellular heterogeneity, such as biocide persistence, can only be tackled by studying each individual in a cell population. Fluorescent tags provide tools for the high-throughput analysis of genomes, RNA transcripts, or proteins on the single-cell level. However, the analysis of lower-molecular-weight compounds that elude tagging is still a great challenge. Here, we describe a novel high-throughput microscale sample preparation technique for single cells that allows a mass spectrum to be obtained for each individual cell within a microbial population. The approach presented includes spotting Chlamydomonas reinhardtii cells, using a noncontact microarrayer, onto a specialized slide and controlled lysis of cells separated on the slide. Throughout the sample preparation, analytes were traced and individual steps optimized using autofluorescence detection of chlorophyll. The lysates of isolated cells are subjected to a direct, label-free analysis using matrix-assisted laser desorption ionization mass spectrometry. Thus, we were able to differentiate individual cells of two Chlamydomonas reinhardtii strains based on single-cell mass spectra. Furthermore, we showed that only population profiles with real single-cell resolution render a nondistorted picture of the phenotypes contained in a population.

High-throughput nucleoside phosphate monitoring in mammalian cell fed-batch cultivation using quantitative matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Current methods for monitoring multiple intracellular metabolite levels in parallel are limited in sample throughput capabilities and analyte selectivity. This article presents a novel high-throughput method based on matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOF-MS) for monitoring intracellular metabolite levels in fed-batch processes. The MALDI-TOF-MS method presented here is based on a new microarray sample target and allows the detection of nucleoside phosphates and various other metabolites using stable isotope labeled internal standards. With short sample preparation steps and thus high sample throughput capabilities, the method is suitable for monitoring mammalian cell cultures, such as antibody producing hybridoma cell lines in industrial environments. The method is capable of reducing the runtime of standard LC-UV methods to approximately 1 min per sample (including 10 technical replicates). Its performance is exemplarily demonstrated in an 8-day monitoring experiment of independently controlled fed-batches, containing an antibody producing mouse hybridoma cell culture. The monitoring profiles clearly confirmed differences between cultivation conditions. Hypothermia and hyperosmolarity were studied in four bioreactors, where hypothermia was found to have a positive effect on the longevity of the cell culture, whereas hyperosmolarity lead to an arrest of cell proliferation. The results are in good agreement with HPLC-UV cross validation experiments. Subsequent principal component analysis (PCA) clearly separates the different bioreactor conditions based on the measured mass spectral profiles. This method is not limited to any cell line and can be applied as a process analytical tool in biotechnological processes.

Rapid estimation of the energy charge from cell lysates using matrix-assisted laser desorption/ionization mass spectrometry: role of in-source fragmentation.

Nucleotides are key players in the central energy metabolism of cells. Here we show how to estimate the energy charge from cell lysates by direct negative ion matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) using 9-aminoacridine as matrix. We found a high level of in-source decay of all the phosphorylated nucleotides, with some of them producing considerable amounts of adenosine-5'-diphosphate (ADP) fragment ions. We investigated the behavior of adenosine-5'-monophosphate (AMP), ADP, and adenosine-5'-triphosphate (ATP) as well as the cofactors coenzyme A (CoA) and acetyl-coenzyme A (ACoA) and nicotinamide adenine dinucleotides (NAD⁺ and NADH) in detail. In-source decay of these compounds depends strongly on the applied laser power and on the extraction pulse delay. At standard instrument settings, the 9-aminoacridine (9-AA) matrix resulted in a much higher in-source decay compared with 2,4,6-trihydroxyacetophenone (2,4,6-THAP). By adding ¹³C-labeled ATP to a cell lysate, we were able to determine the degree of in-source decay during an experiment. Analyzing a cell extract of the monocytic cell line THP-1 with [¹³C]ATP as internal standard, we were able to obtain values for the energy charge that were similar to those determined by a reference liquid chromatography electrospray ionization coupled to mass spectrometry (LC-ESI-MS) method.

Analysis of single algal cells by combining mass spectrometry with Raman and fluorescence mapping.

In order to investigate metabolic properties of single cells of freshwater algae (Haematococcus pluvialis), we implement matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) in combination with microspectroscopic mapping. Straightforward coupling of these two detection platforms was possible thanks to the self-aliquoting properties of micro-arrays for mass spectrometry (MAMS). Following Raman and fluorescence imaging, the isolated cells were covered with a MALDI matrix for targeted metabolic analysis by MALDI-MS. The three consecutive measurements carried out on the same cells yielded complementary information. Using this method, we were able to study the encystment of H. pluvialis - by monitoring the adenosine triphosphate (ATP) to adenosine diphosphate (ADP) ratio during the build-up of astaxanthin in the cells as well as the release of ß-carotene, the precursor of astaxanthin, into the cytosol.

Self-aliquoting microarray plates for accurate quantitative matrix-assisted laser desorption/ionization mass spectrometry.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a fast analysis tool employed for the detection of a broad range of analytes. However, MALDI-MS has a reputation of not being suitable for quantitative analysis. Inhomogeneous analyte/matrix co-crystallization, spot-to-spot inhomogeneity, as well as a typically low number of replicates are the main contributing factors. Here, we present a novel MALDI sample target for quantitative MALDI-MS applications, which addresses the limitations mentioned above. The platform is based on the recently developed microarray for mass spectrometry (MAMS) technology and contains parallel lanes of hydrophilic reservoirs. Samples are not pipetted manually but deposited by dragging one or several sample droplets with a metal sliding device along these lanes. Sample is rapidly and automatically aliquoted into the sample spots due to the interplay of hydrophilic/hydrophobic interactions. With a few microliters of sample, it is possible to aliquot up to 40 replicates within seconds, each aliquot containing just 10 nL. The analyte droplet dries immediately and homogeneously, and consumption of the whole spot during MALDI-MS analysis is typically accomplished within few seconds. We evaluated these sample targets with respect to their suitability for use with different samples and matrices. Furthermore, we tested their application for generating calibration curves of standard peptides with α-cyano-4-hdydroxycinnamic acid as a matrix. For angiotensin II and [Glu(1)]-fibrinopeptide B we achieved coefficients of determination (r(2)) greater than 0.99 without the use of internal standards.

Bispidine platform grants full control over magnetic state of ferrous chelates in water.

A bicyclic ligand platform for iron(II), which allows total control over the complex's magnetic properties in aqueous solution simply by varying one of the six coordination sites of the bispidine ligand, is reported. To achieve this, an efficient synthetic route to an N5 bispidine framework (ligand L4) that features an unsubstituted N-7 site is established. Then, by choosing appropriate N-7-coordinating substituents, the spin state of choice can be imposed on the corresponding ferrous complexes under environmentally relevant conditions in water and near-room temperature. Importantly, the first low-spin and diamagnetic iron(II) chelates in the bispidine series, both in the solid state and in aqueous solution, are reported. The eradication of head-on steric clashes between pendent coordinating arms is at the origin of this success. A new pair of constitutionally similar ferrous coordination compounds of a multidentate ligand system is obtained, which exhibits a distinctly binary (off-on) magnetic relationship. The new synthetic intermediate L4 may be substituted in just one step by any desired pendent arm, thus allowing access to complexes with finely tuned magnetic properties.

1,2:3,4-Diepoxybutane in blood of male B6C3F1 mice and male Sprague-Dawley rats exposed to 1,3-butadiene.

The important industrial chemical 1,3-butadiene (BD; CAS Registry Number: 106-99-0) is a potent carcinogen in B6C3F1 mice and a weak one in Sprague-Dawley rats. This difference is mainly attributed to the species-specific burden by the metabolically formed 1,2:3,4-diepoxybutane (DEB). However, only limited data exist on the DEB blood burden of rodents at BD concentrations below 100 ppm. Considering this, DEB concentrations were determined in the blood of mice and rats immediately after 6h exposures to various constant concentrations of BD of between about 1 and 1200 ppm. Immediately after its collection, blood was injected into a vial that contained perdeuterated DEB (DEB-D(6)) as internal standard. Plasma samples were prepared and treated with sodium diethyldithiocarbamate that derivatized metabolically produced DEB and DEB-D(6) to their bis(dithiocarbamoyl) esters, which were then analyzed by high performance liquid chromatography coupled with an electrospray ionization tandem mass spectrometer. DEB concentrations in blood versus BD exposure concentrations in air could be described by one-phase exponential association functions. Herewith calculated (±)-DEB concentrations in blood increased in mice from 5.4 nmol/l at 1 ppm BD to 1860 nmol/l at 1250 ppm BD and in rats from 1.2 nmol/l at 1 ppm BD to 92 nmol/l at 200 ppm BD, at which exposure concentration 91% of the calculated DEB plateau concentration in rat blood was reached. This information on the species-specific blood burden by the highly mutagenic DEB helps to explain why the carcinogenic potency of BD in rats is low compared to that in mice.