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.

Single-cell mass spectrometry reveals the importance of genetic diversity and plasticity for phenotypic variation in nitrogen-limited Chlamydomonas.

Phenotypic variation is vital for microbial populations to survive environmental perturbations. Both genetic and non-genetic factors contribute to an organism's phenotypic variation and therefore its fitness. To investigate the correlation between genetic diversity and phenotypic variation, we applied our recently developed mass spectrometry method that allows for the simultaneous measurement of more than 25 different lipids and pigments with high throughput in the unicellular microalga Chlamydomonas reinhardtii. We monitored the impact of nitrogen limitation on a genetically diverse wild-type strain CC-1690 and two isoclonal isolates from CC-1690 named ANC3 and ANC5. Measuring molecular composition of thousands of single cells at different time points of the experiment allowed us to capture a dynamic picture of the phenotypic composition and adaptation of the populations over time. Although the genetically diverse population maintained phenotypic variation over the whole time course of the experiment, the isoclonal cultures showed higher synchronicity in their phenotypic response. Furthermore, the genetically diverse population showed equal or greater phenotypic variation over the whole time range in multidimensional trait space compared with isoclonal populations. However, along individual trait axes non-genetic variance was higher in isoclonal populations.

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.

A Microarray-Matrix-assisted Laser Desorption/Ionization-Mass Spectrometry Approach for Site-specific Protein N-glycosylation Analysis, as Demonstrated for Human Serum Immunoglobulin M (IgM).

We demonstrate a new approach for the site-specific identification and characterization of protein N-glycosylation. It is based on a nano-liquid chromatography microarray-matrix assisted laser desorption/ionization-MS platform, which employs droplet microfluidics for on-plate nanoliter reactions. A chromatographic separation of a proteolytic digest is deposited at a high frequency on the microarray. In this way, a short separation run is archived into thousands of nanoliter reaction cavities, and chromatographic peaks are spread over multiple array spots. After fractionation, each other spot is treated with PNGaseF to generate two correlated traces within one run, one with treated spots where glycans are enzymatically released from the peptides, and one containing the intact glycopeptides. Mining for distinct glycosites is performed by searching for the predicted deglycosylated peptides in the treated trace. An identified peptide then leads directly to the position of the "intact" glycopeptide clusters, which are located in the adjacent spots. Furthermore, the deglycosylated peptide can be sequenced efficiently in a simple collision-induced dissociation-MS experiment. We applied the microarray approach to a detailed site-specific glycosylation analysis of human serum IgM. By scanning the treated spots with low-resolution matrix assisted laser desorption/ionization-time-of-flight-MS, we observed all five deglycosylated peptides, including the one originating from the secretory chain. A detailed glycopeptide characterization was then accomplished on the adjacent, untreated spots with high mass resolution and high mass accuracy using a matrix assisted laser desorption ionization-Fourier transform-MS. We present the first detailed and comprehensive mass spectrometric analysis on the glycopeptide level for human polyclonal IgM with high mass accuracy. Besides complex type glycans on Asn 395, 332, 171, and on the J chain, we observed oligomannosidic glycans on Asn 563, Asn 402 and minor amounts of oligomannosidic glycans on the glycosite Asn 171. Furthermore, hybrid type glycans were found on Asn 402, Asn 171 and in traces Asn 332.

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.

Quantification of saquinavir from lysates of peripheral blood mononuclear cells using microarrays and standard MALDI-TOF-MS.

Drug monitoring is usually performed by liquid chromatography coupled with optical detection or electrospray ionization mass spectrometry. More recently, matrix-assisted laser desorption/ionization (MALDI) in combination with triple quadrupole or Fourier-transform (FT) mass analyzers has also been reported to allow accurate quantification. Here, we present a strategy that employs standard MALDI time-of-flight (TOF) mass spectrometry (MS) for the sensitive and accurate quantification of saquinavir from an extract of blood peripheral mononuclear cells. Unambiguous identification of saquinavir in the mass spectra was possible because of using internal mass calibration and by an overall low chemical noise in the low mass range. Exact mass determination of the constant background peaks of the cell extract, which were used for recalibration, was performed by an initial MALDI-FT-MS analysis. Fast and multiplexed sample analysis was enabled by microarray technology, which provided 10 replicates in the lower nL range for each sample in parallel lanes on a chip. In order to validate the method, we employed various statistical tests, such as confidence intervals for linear regressions, three quality control samples, and inverse confidence limits of the estimated concentration ratios.

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.

Fate and transformation of silver nanoparticles in urban wastewater systems.

Discharge of silver nanoparticles (Ag-NP) from textiles and cosmetics, todays major application areas for metallic Ag-NP, into wastewater is inevitable. Transformation and removal processes in sewers and wastewater treatment plants (WWTP) will determine the impact of Ag-NP on aquatic and terrestrial environments, via the effluents of the WWTP and via the use of digested sludge as fertilizer. We thus conducted experiments addressing the behavior of Ag-NP in sewers and in WWTP. We spiked Ag-NP to a 5 km long main trunk sewer and collected 40 wastewater samples after 500 m, 2400 m and 5000 m each according to the expected travel times of the Ag-NP. Excellent mass closure of the Ag derived by multiplying the measured Ag concentrations times the volumetric flow rates indicate an efficient transport of the Ag-NP without substantial losses to the sewer biofilm. Ag-NP reacted with raw wastewater in batch experiments were sulfidized to roughly 15% after 5 h reaction time as revealed by X-ray absorption spectroscopy (XAS). However, acid volatile sulfide (AVS) concentrations were substantially higher in the sewer channel (100 µM) compared to the batch experiments (3 µM; still sufficient to sulfidize spiked 2 µM Ag) possibly resulting in a higher degree of sulfidation in the sewer channel. We further investigated the removal efficiency of 10 nm and 100 nm Ag- and gold (Au)-NP coated with citrate or polyvinylpyrrolidone in activated sludge batch experiments. We obtained very high removal efficiencies (≈ 99%) irrespective of size and coating for Ag- and Au-NP, the latter confirming that the particle type was of minor importance with respect to the degree of NP removal. We observed a strong size dependence of the sulfidation kinetics. We conclude that Ag-NP discharged to the wastewater stream will become sulfidized to various degrees in the sewer system and are efficiently transported to the WWTP. The sulfidation of the Ag-NP will continue in the WWTP, but primarily depending on the size the Ag-NP, may not be complete. Very high removal efficiencies in the WWTP will divert most of the Ag-NP mass flow to the digester and only a small fraction of the Ag will be released to surface waters.