Using Dicationic Ion-Pairing Compounds To Enhance the Single Cell Mass Spectrometry Analysis Using the Single-Probe: A Microscale Sampling and Ionization Device

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 single-probe, 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, 3-propanediyl-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 the 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.

microMS: A Python Platform for Image-Guided Mass Spectrometry Profiling

“…The design of the software architecture simplifies the addition of new instruments. The integration of ambient ionization methods, including single-probe [20–22] or nanospray desorption electrospray ionization [23], are enticing candidates as they have demonstrated single cell sensitivities in imaging and profiling applications. Currently, four specific instrument implementations are supplied in the coordinateMappers package: a Bruker UltrafleXtreme, a Bruker solariX, the AB Sciex oMALDI sample stage attached to a custom hybrid MALDI/C 60 + -SIMS, and a lab-built 3-axis liquid microjunction probe.…”

Review and perspectives on the applications of mass spectrometry imaging under ambient conditions

“…In contrast, higher signal intensities of the same lipid‐dication pairs were found with [C 5 (bpyr) 2 F 2 ] than with [C 3 (triprp) 2 F 2 ]; the shorter linker chain between the two nitrogen charged centres in [C 3 (triprp) 2 F 2 ] may sterically hinder lipid‐dication formations and offer selective preference for small molecule binding as opposed to large biomolecule (e.g. lipid) binding . The reactivity and selectivity of dicationic salts specifically to PE, PS, PG and PI are not entirely clear, yet these studies illustrate the importance of appropriately selecting reactive agents to improve molecular coverage for targeted MS imaging analyses.…”

“…Rao and coworkers detected 526 and 322 negatively charged species in positive ion mode by using the dicationic salts 1,5‐pentanediylbis (1‐butylpyrrolidinium) difluoride, [C 5 (bpyr) 2 F 2 ] and 1,3‐propanediyl‐bis (tripropylphosphonium) difluoride, [C 3 (triprp) 2 F 2 ], respectively, in the SP‐MS imaging of rat brain tissues . Spatially resolved images of 17μm of lipid‐dication pairs of docosahexaenoic acid (22:6), phosphatidic acid (PA) (36:1), PE (P‐38:6), PG (34:1), PS (40:6) and PI (38:4) with [C 5 (bpyr) 2 F 2 ] and [C 3 (triprp) 2 F 2 ] were visualized.…”

“…Recent developments are being directed towards single cells for the characterization and visualization of cellular and subcellular biological metabolomics in real time ambient MS analysis . Single cell analysis has been capable via Single Probe MS of the sampling and ionization of intracellular constituents of live single eukaryotic cells and cervical cancer HeLa cells with a single‐probe tip of 6–10 μm, a diameter small enough for the tip to be inserted into individual live cells.…”

Graphene Coated Nanoprobes: A Review

“…Sharp probe tips with an apex radius <100 nm are widely used in many different fields of science including electronics [1], physics [2], chemistry [3], biology [4] and medicine [5], as they allow local characterization and manipulation of materials with a nanometric spatial resolution. Depending on the equipment and application in which they are used, nanoprobes can have a wide variety of shapes, material composition and prizes.…”