Development of Pico-ESI-MS for Single-Cell Metabolomics Analysis.

In this chapter, we introduced a Pico-ESI strategy for metabolomics analysis with picoliter-level samples. This Pico-ESI strategy was technically achieved by pulsed direct current electrospray ionization source (Pulsed-DC-ESI). This source could collect MS signals for a few minutes from a cell, enabling us to obtain large-scale MS2 data of metabolite IDs in single-cell analysis. Further identification of the single-cell metabolome such as the database match and chemical modification to metabolome was thereby achieved. Technically, this source could ionize sample with no need of sample and electrode contact, which can be potentially applied for high-throughput analysis. We also introduced several strategies related to Pico-ESI to reduce the matrix interference especially for extremely small samples developed in our group, including step-voltage nanoelectrospray, picoliter sample desalting method, droplet-based microextraction method, and probe-ESI, etc. All these strategies have been successfully applied to single-cell analysis.

Detecting Low-Abundance Molecules at Single-Cell Level by Repeated Ion Accumulation in Ion Trap Mass Spectrometer.

Low-abundance metabolites or proteins in single-cell samples are usually undetectable by mass spectrometry (MS) due to the limited amount of substances in single cells. This limitation inspired us to further enhance the sensitivity of commercial mass spectrometers. Herein, we developed a technique named repeated ion accumulation by ion trap MS, which is capable of enhancing the sensitivity by selectively and repeatedly accumulating ions in a linear ion trap for up to 25 cycles. The increase in MS sensitivity was positively correlated with the number of repeated cycles. When ions were repeatedly accumulated for 25 cycles, the sensitivity of adenosine triphosphate detection was increased by 22-fold within 1.8 s. Our technique could stably detect low-abundance ions, especially MSn ions, at the single-cell level, such as 5-methylcytosine hydrolyzed from sample equivalent to ∼0.2 MCF7 cell. The strategy presented in this study offers the possibility to aid single-cell analysis by enhancing MS detection sensitivity.

Integrated Droplet-Based Microextraction with ESI-MS for Removal of Matrix Interference in Single-Cell Analysis.

Integrating droplet-based microfluidics with mass spectrometry is essential to high-throughput and multiple analysis of single cells. Nevertheless, matrix effects such as the interference of culture medium and intracellular components influence the sensitivity and the accuracy of results in single-cell analysis. To resolve this problem, we developed a method that integrated droplet-based microextraction with single-cell mass spectrometry. Specific extraction solvent was used to selectively obtain intracellular components of interest and remove interference of other components. Using this method, UDP-Glc-NAc, GSH, GSSG, AMP, ADP and ATP were successfully detected in single MCF-7 cells. We also applied the method to study the change of unicellular metabolites in the biological process of dysfunctional oxidative phosphorylation. The method could not only realize matrix-free, selective and sensitive detection of metabolites in single cells, but also have the capability for reliable and high-throughput single-cell analysis.

Pulsed Direct Current Electrospray: Enabling Systematic Analysis of Small Volume Sample by Boosting Sample Economy.

We had developed pulsed direct current electrospray ionization mass spectrometry (pulsed-dc-ESI-MS) for systematically profiling and determining components in small volume sample. Pulsed-dc-ESI utilized constant high voltage to induce the generation of single polarity pulsed electrospray remotely. This method had significantly boosted the sample economy, so as to obtain several minutes MS signal duration from merely picoliter volume sample. The elongated MS signal duration enable us to collect abundant MS(2) information on interested components in a small volume sample for systematical analysis. This method had been successfully applied for single cell metabolomics analysis. We had obtained 2-D profile of metabolites (including exact mass and MS(2) data) from single plant and mammalian cell, concerning 1034 components and 656 components for Allium cepa and HeLa cells, respectively. Further identification had found 162 compounds and 28 different modification groups of 141 saccharides in a single Allium cepa cell, indicating pulsed-dc-ESI a powerful tool for small volume sample systematical analysis.

DNA methylation as a potential diagnosis indicator for rapid discrimination of rare cancer cells and normal cells.

The global DNA methylation degree may be a ubiquitous and early biomarker to distinguish cancer cells from benign cells. However, its usefulness in clinical diagnosis was scarcely demonstrated, because the cancer cells isolated from patients were usually very rare. Even if 10 mL of peripheral blood was sampled from a patient, only tens of cancer cells could be isolated. So a method to quantify DNA methylation from small number of cells was needed to apply DNA methylation in clinical environment. In this study, we found that normal breast cell line MCF10A and breast cancer cell line MCF7 cells present significantly different percentage of genomic 5-methylcytosine (p < 0.02, n = 8), it could be a potential indicator for rapid discrimination of rare cancer cells from normal cells. However, conventional mass spectrometry needs usually ~10(6) cells to quantify DNA methylation degree, which was too large to be applied in clinical diagnosis. Here we developed a fast mass spectrometry-based method capable of analyzing the DNA methylation degree from only ~100 human cells. Our method could reveal the different DNA methylation degree between MCF10A and MCF7 cells in less than two hours, having the potential to provide reliable information for clinical application.

Coupling a solid phase microextraction (SPME) probe with ambient MS for rapid enrichment and detection of phosphopeptides in biological samples.

In this study, we developed a probe-electrospray ionization method by coupling a SPME probe modified with nanosized TiO2 directly to nanoESI-MS for the phosphoproteome analysis, which demonstrated excellent selectivity and sensitivity for enrichment of phosphopeptides in complex biological samples.