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Using Dicationic Ion-Pairing Compounds to Enhance the Single Cell Mass Spectrometry Analysis using the Singleprobe: A Microscale Sampling and Ionization Device Ning Pan, Wei Rao, Shawna J. Standke, and Zhibo Yang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b01284 • Publication Date (Web): 30 May 2016 Downloaded from http://pubs.acs.org on June 3, 2016
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Analytical Chemistry
Using Dicationic Ion-Pairing Compounds to Enhance the Single Cell Mass Spectrometry Analysis using the Single-probe: A Microscale Sampling and Ionization Device
AUTHOR NAMES
Ning Pan†, Wei Rao†, Shawna J. Standke, Zhibo Yang*
AUTHOR ADDRESS Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma 73019, USA †
These authors contributed equally to this work.
*Address reprint requests to Dr. Zhibo Yang, Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma 73019, USA Tel: (405) 325-1772
Email:
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Abstract A unique mass spectrometry (MS) method has been developed to determine the negatively charged species in live single cells using the positive ionization mode. The method utilizes dicationic ion-pairing compounds through the miniaturized multifunctional device, the Singleprobe, for reactive MS analysis of live single cells under ambient conditions. In this study, two dicationic reagents, 1,5-Pentanediyl-bis(1-butylpyrrolidinium) difluoride (C5(bpyr)2F2) and 1,3Propanediyl-bis(tripropylphosphonium) difluoride (C3(triprp)2F2), were added in the solvent and introduced into single cells to extract cellular contents for real-time MS analysis. The negatively charged (1− charged) cell metabolites, which form stable ion-pairs (1+ charged) with dicationic compounds (2+ charged), were detected in positive ionization mode with a greatly improved sensitivity. We have tentatively assigned 192 and 70 negatively charged common metabolites as adducts with (C5(bpyr)2F2) and (C3(triprp)2F2), respectively, in three separate SCMS experiments in the positive ion mode. The total number of tentatively assigned metabolites is 285 for C5(bpyr)2F2 and 143 for C3(triprp)2F2. In addition, the selectivity of dicationic compounds in the complex formation allows for the discrimination of overlapped ion peaks with low abundances. Tandem (MS/MS) analyses at single cell level were conducted for selected adduct ions for molecular identification. The utilization of the dicationic compounds in the Single-probe MS technique provides an effective approach to the detection of a broad range of metabolites at the single cell level.
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INTRODUCTION Single cell analysis (SCA) has become an important and increasingly active area in biological studies. Compared with the traditional methods that are based on the population averaged studies, SCA can provide a more nuanced analysis of the underlying biological mechanics of the system being studied by illustrating biological differences at the level of individual cells transcriptomics
1-4
5-8
. SCA encompasses a variety of analytical techniques, including single cell , single cell genomics9, single cell fluorescent tagging 10, Raman spectroscopy
imaging 11 and others. Single cell mass spectrometry (SCMS) is a nascent field that has gained a great interest in mass spectrometry (MS) research
12-14
. MS is a versatile technique to simultaneously analyze a
large number of molecules in a short period of time. Traditional MS approaches to cell analysis were restricted to a population of cells (such as cell lysate), where an averaged result is obtained. Recent advancements in high mass resolution MS has allowed for the confident assignment of large numbers of molecules
15
, and improved sensitivity enables MS to be applied at the single
cell level, mostly in the field of metabolomics, proteomics
17
14
and potentially single cell peptidomics
16
and
. Current SCMS techniques can be broadly categorized into non-ambient and
ambient techniques, based on their sampling environment. Common non-ambient techniques include matrix assisted laser desorption/ionization (MALDI) MS secondary ion MS (TOF-SIMS)
20-21
18-19
and time-of-flight
approaches, which are capable of high spatial resolution
22
for cellular and sub-cellular resolution analysis of the cell organelles 23-25. However, non-ambient techniques require obligatory sample pretreatment and a vacuum sampling environment, and they are not suitable for live cell analysis.
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Ambient SCMS techniques allow for analysis of live single cells in ambient environment with little to no sample preparation. Techniques include laser assisted electrospray ionization 26-27
(LAESI) MS
, single cell capillary electrophoresis (CE) ESI MS
28-29
, probe ESI MS
30
, and
live single-cell video-MS (live MS)31-33. Among these methods, the live MS technique is based on the direct extraction of cellular compounds using a sharp nano-ESI emitter followed by offline MS analysis
32
. However, due to the separate steps needed for sampling and analysis in live MS
experiment, metabolites that are sensitive to the cell status are possibly changed during sample transfer and preparation. Recently, we have introduced the Single-probe MS technique for in situ MS analysis of live eukaryotic cells in real-time 34-35. The Single-probe is an integrated micro-scale sampling and ionization device that can be coupled with MS for multiple applications. The tip (6~10 μm) of the Single-probe can be directly inserted into single cells for direct liquid-extraction of cellular contents followed by immediate MS detection. This technique has been used to analyze cellular metabolites of single cancer cells treated with drugs 35. In addition, the Single-probe can be used for other applications, including high spatial resolution ambient MS imaging of biological samples 34, 36. In general, the ionizable cellular metabolites are detected either as cations or anions, and experiments are sometimes conducted in both ionization modes to improve detection coverage
37
.
However, this type of analysis can be challenging for SCMS due to the extremely small amount of cellular content that is available from a cell (typically ~1 picoliter)
1, 31
, making repeated
analyses of the same cell an impractical procedure. To maximize the amount of information from a SCMS experiment, we have developed the reactive Single-probe SCMS method, in which dicationic compounds are used as reagents to improve the detection coverage and the number of
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molecular assignments. Dicationic compounds, sometimes referred as ionic liquids
38
, are
synthetic molecules with two positive charges (2+ charged) that are readily form ion-paring adducts with a wide range of anions (1– charged), such that the adducts (overall 1+ charged) can be detected in positive ionization mode MS 39. Dicationic compounds have been successfully used in electrospray ionization (ESI) MS (i.e., paired ion ESI (PIESI)), where a variety of negatively charged species, such as phospholipids
40
and pesticides
41
, were detected in the positive
ionization mode, with the increased ion signal intensity compared with the negative ionization mode. Dicationic compounds were also used in desorption electrospray ionization (DESI) MS for the detection of phospholipids in the positive ionization mode 42, where one dicationic compound can selectively enhance the detection sensitivity of relatively small lipids (< 400 m/z). These dicationic compounds were later used for DESI MS imaging of biological tissue sections, and the MS images of a number of negatively charged species were obtained in the positive ion mode43. Recently,
two
commercially
butylpyrrolidinium)
difluoride
available
dicationic
(C5(bpyr)2F2,
compounds,
Figure
1a)
1,5-Pentanediyl-bis(1and
1,3-Propanediyl-
bis(tripropylphosphonium) difluoride (C3(triprp)2F2, Figure 1b), were used in a Single-probe ambient MS imaging study of mouse brain sections to produce high spatial and mass resolution MS images
44
. This study indicates that both dicationic compounds readily formed positively
charged adducts with a broad range of negatively charged metabolites, allowing MS images of a much larger number of metabolites to be produced from a single experiment than previously possible. Here, we used these two dicationic compounds, C5(bpyr)2F2 and C3(triprp)2F2, as the reagents to enable the detection of negatively charged metabolites in the positive ionization mode for real-time SCMS analysis. The dicationic compounds were added into the sampling solvent,
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and introduced into individual cells using the Single-probe SCMS setup (Figures 2 and S1). We were able to detect a significantly larger number of metabolites when using C5(bpyr)2F2 than using conventional positive or negative ionization modes alone. In particular, this reactive SCMS approach provides higher ion signal intensities for the adducted metabolites. Using a Thermo Orbitrap XL mass spectrometer and the home-built Single-probe SCMS setup
35
controlled using
the LabView software package 45, high mass accuracy MS analyses were performed for tentative molecular assignments (mass error
