Robert F. Steinhoff

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 β-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.

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.

A new strategy based on real-time secondary electrospray ionization and high-resolution mass spectrometry to discriminate endogenous and exogenous compounds in exhaled breath

Breath is considered to be an easily accessible matrix, whose chemical composition relates to compounds present in blood. Therefore many metabolites are expected in exhaled breath, which may be used in the future for the development of diagnostic methods. In this article, a new strategy to discriminate between exhaled endogenous metabolites and exhaled exogenous contaminants by direct high-resolution mass spectrometry is introduced. The analysis of breath in real-time by secondary electrospray ionization mass spectrometry allows to interpret the origin of exhaled compounds. Exhaled metabolites that originate in the respiratory system show reproducible and significant patterns if plotted in real-time (>1 data point per second). An exhaled metabolite shows a signal that tends to rise at the end of a complete (forced) exhalation. In contrast, exogenous compounds, which may be present in room air, are gradually diluted by the air from the deeper lung and therefore show a trend of falling intensity. Signals found in breath by using this pattern recognition are linked to potential metabolites by comparison with online databases. In addition to this real-time approach, it is also shown how to combine this method with classical analytical methods in order to potentially identify unknown metabolites. Finally exhaled compounds following smoking a cigarette, chewing gum, or drinking coffee were investigated to underline the usefulness of this new approach.

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

ABSTRACT 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 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.

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.

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.

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.

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.