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Quantitative Extraction and Mass Spectrometry Analysis at a Single-Cell Level
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posted on 2018-06-06, 00:00 authored by Ruichuan Yin, Venkateshkumar Prabhakaran, Julia LaskinQuantitative live
cell mass spectrometry analysis at a subcellular
level requires the precisely controlled extraction of subpicoliter
volumes of material from the cell, sensitive analysis of the extracted
analytes, and their accurate quantification without prior separation.
In this study, we demonstrate that localized electroosmotic extraction
provides a direct path to addressing this challenge. Specifically,
we demonstrate quantitative mass spectrometry analysis of biomolecules
in picoliter volumes extracted from live cells. Electroosmotic extraction
was performed using two electrodes and a finely pulled nanopipette
with tip diameter of <1 μm containing a hydrophobic electrolyte
compatible with mass spectrometry analysis. The electroosmotic drag
was used to drive analytes out of the cell into the nanopipette. Analyte
molecules extracted both from solutions and cell samples were analyzed
using nanoelectrospray ionization (nanoESI) directly from the nanopipette
into a mass spectrometer. More than 50 metabolites including sugars
and flavonoids were detected in positive mode in 2−5 pL volumes
of the cytoplasmic material extracted from Allium cepa. Quantification of the extracted glucose was performed using sequential
extraction of a known volume of the aqueous solution containing glucose-d2 standard of known concentration. We found
that the ratio of the signal of glucose to glucose-d2 increased linearly with glucose concentration. This
observation indicates that the approach developed in this study enables
quantitative analysis of small volumes of metabolites extracted from
cells. Furthermore, we observed efficient separation of hydrophilic
and hydrophobic analytes through partitioning into the aqueous and
hydrophobic electrolyte phase, respectively, which provides additional
important information on the molecular properties of extracted metabolites.
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electrolyte phasesubpicoliter volumescell samplesmass spectrometry analysisnanoelectrospray ionizationQuantitative ExtractionElectroosmotic extractionsubcellular leveltip diameternanopipetteAllium cepaglucose concentrationAnalyte moleculespicoliter volumescell mass spectrometry analysisMass Spectrometry AnalysisSingle-Cell Level Quantitativesequential extraction50 metaboliteselectroosmotic dragmass spectrometercytoplasmic materialelectroosmotic extractiondrive analytes
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