A MALDI-based Chip Technology for Single-Cell Mass Spectrometry (SCMS)

Renato Zenobi, Fagerer Stephan, Martin Pabst, Jens Boertz

Analytix 2014, Issue 1

Heterogeneity is a characteristic attribute of all populations of living organisms. Cell-to-cell variability can be due to a number of reasons; e.g., non-uniformity of the cellular environment, such as a given cell within the cell cycle, genetic differences resulting from mutations or stochastic events in gene expression. However, examination of the underpinnings of intrapopulation variability of cells can only be achieved with single-cell analysis.

Analysis of tiny quantities of small molecules in complex mixtures/matrices is a challenge. Fluorometric approaches generally need the introduction of fluorescent tags; thus, their application to studies of low-molecular-weight compounds in cells and cell extracts is limited to a small number of analytes. Mass spectrometry (MS) is a label-free analytical technique which permits identification of virtually any analyte as well as structural analysis.

During the past decades, the application of mass spectrometry for the analysis of small molecules increased, and MS became the key technique in metabolomics.

Single-cell mass spectrometry (SCMS) is a rapidly growing discipline of analytical chemistry, and many examples of its use for the analysis of cells can be found. A few interesting studies show MS analysis of a couple of metabolites present in single cells. An example of a SCMS study by electrospray ionization (ESI) is the work by Mizuno et al., in which mass spectra corresponding to individual cells obtained from different cell lines were classified. Applications of SCMS based on matrix-assisted laser desorption/ionization (MALDI) for studying biomolecules in single neurons have also been shown.

MALDI-MS is often used in proteomics; however, its potential in metabolomics has recently been demonstrated, as well. MALDI-MS can provide efficient use of the limited amounts of analytes, and its sensitivity with respect to primary metabolites, such as nucleotides is adequate to detect the small levels present in single cells. In addition, MALDI-MS is less dependent on sample matrix composition than other techniques such as ESI-MS.

Here, We presenting the new MAMS for single-cell analysis. A few products used for the first successful application of the MAMS target are listed in Table 1.

Table 1. Sigma-Aldrich products used for single-cell MALDI-MS

Cat. No.	Material	Application Area	Package Size
50757	MAMS for single-cell mass spectrometry	Single-cell MS	1 slide
92817	9-Aminoacridine	MALDI-MS	1 g

Size of MAMS

The MAMS has the dimensions of a standard microscope slide containing two areas of 13x13 spots of 100 µM diameter (in total, 338 spots per slide)

Compatibility of MAMS

Experiments were done using a 4800 Plus MALDI TOF/TOF™ analyzer and a TOF/TOF 5800, (both ABsciex, see references). The microarrays were also tested on a Bruker Ultraflex Extreme MALDI Tandem TOF Mass Spectrometer. Please keep in mind that additional adapters for the MAMS slides are needed, e.g., LaserBio Labs Mass Spectrometry Imaging Starter Kit (AB) or Bruker Daltonik MTP Slide Adapter II for the Bruker Ultraflex series. Spot set designs/geometry files, for both systems (AB Sciex and Bruker MALDI instruments) can be supplied on request by the instrument manufacturers.

Aliquoting Process

Briefly, cells are taken from a liquid culture, quenched using cold solvents (to stop any metabolic activity), after which the supernatant is removed and cells are washed to remove salts. The cell suspension is then spread onto the MAMS target plate. Applying the cell suspension onto the MAMS surface will result in an automated aliquoting of the cell suspension into the hydrophilic reservoirs, without the need for a microspotter.

Depending on the cell concentration used, the number of cells on each hydrophilic reservoir can be between zero and hundreds. The transparency of the MAMS substrate allows for microscopic analysis to determine the number of cells in each reservoir while the cellular metabolism remains quenched because the entire MAMS chip is kept cold in a cryochamber.

9-AA MALDI matrix can be applied by spraying, as described in the references below.

Figure 1. Image of the single cell micro-array square of 13 x 13 spots. Each spot has a diameter of 100 µM. Two arrays are placed on an omniphobic coated ITO slide in dimensions of a standard microscope slide.

References

1. Amantonico A, Urban PL, Fagerer SR, Balabin RM, Zenobi R. 2010. Single-Cell MALDI-MS as an Analytical Tool for Studying Intrapopulation Metabolic Heterogeneity of Unicellular Organisms. Anal. Chem.. 82(17):7394-7400. https://doi.org/10.1021/ac1015326

2. Ibanez AJ, Fagerer SR, Schmidt AM, Urban PL, Jefimovs K, Geiger P, Dechant R, Heinemann M, Zenobi R. 2013. Mass spectrometry-based metabolomics of single yeast cells. Proceedings of the National Academy of Sciences. 110(22):8790-8794. https://doi.org/10.1073/pnas.1209302110

3. Zenobi R. 2013. Single-Cell Metabolomics: Analytical and Biological Perspectives. Science. 342(6163):1243259-1243259. https://doi.org/10.1126/science.1243259

MALDI TOF/TOF is a trademark of AB Sciex PTE. LTD.