Enhanced sensitivity and metabolite coverage with remote laser ablation electrospray ionization-mass spectrometry aided by coaxial plume and gas dynamics.

Laser ablation electrospray ionization-mass spectrometry (LAESI-MS) allows for direct analysis of biological tissues at atmospheric pressure with minimal to no sample preparation. In LAESI, a mid-IR laser beam (λ = 2.94 µm) is focused onto the sample to produce an ablation plume that is intercepted and ionized by an electrospray at the inlet of the mass spectrometer. In the remote LAESI platform, the ablation process is removed from the mass spectrometer inlet and takes place in an ablation chamber, allowing for incorporation of additional optics for microscopic imaging and targeting of specific features of the sample for laser ablation sampling. The ablated material is transported by a carrier gas through a length of tubing, delivering it to the MS inlet where it is intercepted and ionized by an electrospray. Previous proof-of-principle studies used a prolate spheroid ablation chamber with the carrier gas flow perpendicular to the ablation plume. This design resulted in significant losses of MS signal in comparison to conventional LAESI. Here we present a newly designed conical inner volume ablation chamber that radially confines the ablation plume produced in transmission geometry. The carrier gas flow and the expanding ablation plume are aligned in a coaxial configuration to improve the transfer of ablated particles. This new design not only recovered the losses observed with the prolate spheroid chamber design, but was found to provide an ∼12-15% increase in the number of metabolite peaks detected from plant leaves and tissue sections relative to conventional LAESI.

Remote laser ablation electrospray ionization mass spectrometry for non-proximate analysis of biological tissues.

RATIONALE: We introduce remote laser ablation electrospray ionization (LAESI), a novel, non-proximate ambient sampling technique. Remote LAESI allows additional analytical instrumentation to be incorporated during sample analysis. This work demonstrates the utility of remote LAESI and, when combined with optical microscopy, allows for the microscopy-guided sampling of biological tissues. METHODS: Rapid prototyping using a 3D printer was applied to produce various ablation chamber geometries. A focused 5 ns, 2.94 µm laser pulse kept at 10 Hz ablated the sample within the chamber, remote to the mass spectrometer inlet. Ablated particulates were carried through a transfer tube by N2 gas, delivered to the electrospray plume and ionized. A long-distance microscope was used to capture images of tissues before, during and after ablation. RESULTS: Optimized remote LAESI was found to have a 27% transport efficiency compared with conventional LAESI, sufficient for many applications. A comparable molecular coverage was obtained with remote LAESI for the analysis of plant tissue. Proof-of-principle experiments using a pansy flower and a maple leaf indicated the functionality of this approach for selecting domains of interest for analysis by optical microscopy and obtaining chemical information from those selected regions by remote LAESI-MS. CONCLUSIONS: Remote LAESI is an ambient non-proximate sampling technique, proven to detect metabolites in biological tissues. When combined with optical microscopy, remote LAESI allows for the simultaneous acquisition of morphological and chemical information. This technique has important implications for histology, where chemical information for specific locations within a tissue is critical.

Subcellular metabolite and lipid analysis of Xenopus laevis eggs by LAESI mass spectrometry.

Xenopus laevis eggs are used as a biological model system for studying fertilization and early embryonic development in vertebrates. Most methods used for their molecular analysis require elaborate sample preparation including separate protocols for the water soluble and lipid components. In this study, laser ablation electrospray ionization (LAESI), an ambient ionization technique, was used for direct mass spectrometric analysis of X. laevis eggs and early stage embryos up to five cleavage cycles. Single unfertilized and fertilized eggs, their animal and vegetal poles, and embryos through the 32-cell stage were analyzed. Fifty two small metabolite ions, including glutathione, GABA and amino acids, as well as numerous lipids including 14 fatty acids, 13 lysophosphatidylcholines, 36 phosphatidylcholines and 29 triacylglycerols were putatively identified. Additionally, some proteins, for example thymosin ß4 (Xen), were also detected. On the subcellular level, the lipid profiles were found to differ between the animal and vegetal poles of the eggs. Radial profiling revealed profound compositional differences between the jelly coat vitelline/plasma membrane and egg cytoplasm. Changes in the metabolic profile of the egg following fertilization, e.g., the decline of polyamine content with the development of the embryo were observed using LAESI-MS. This approach enables the exploration of metabolic and lipid changes during the early stages of embryogenesis.

Top-down mass spectrometry imaging of intact proteins by laser ablation ESI FT-ICR MS.

Laser ablation ESI (LAESI) is a recent development in MS imaging. It has been shown that lipids and small metabolites can be imaged in various samples such as plant material, tissue sections or bacterial colonies without any sample pretreatment. Further, LAESI has been shown to produce multiply charged protein ions from liquids or solid surfaces. This presents a means to address one of the biggest challenges in MS imaging; the identification of proteins directly from biological tissue surfaces. Such identification is hindered by the lack of multiply charged proteins in common MALDI ion sources and the difficulty of performing tandem MS on such large, singly charged ions. We present here top-down identification of intact proteins from tissue with a LAESI ion source combined with a hybrid ion-trap FT-ICR mass spectrometer. The performance of the system was first tested with a standard protein with electron capture dissociation and infrared multiphoton dissociation fragmentation to prove the viability of LAESI FT-ICR for top-down proteomics. Finally, the imaging of a tissue section was performed, where a number of intact proteins were measured and the hemoglobin α chain was identified directly from tissue using CID and infrared multiphoton dissociation fragmentation.

A study of electrospray ionization emitters with differing geometries with respect to flow rate and electrospray voltage.

The performance of several electrospray ionization emitters with different orifice inside diameters (i.d.s), geometries, and materials are compared. The sample solution is delivered by pressure driven flow, and the electrospray ionization voltage and flow rate are varied systematically for each emitter investigated, while the signal intensity of a standard is measured. The emitters investigated include a series of emitters with a tapered outside diameters (o.d.) and unaltered i.d.s, a series of emitters with tapered o.d.s and i.d.s, an emitter with a monolithic frit and a tapered o.d., and an emitter fabricated from polypropylene. The results show that for the externally etched emitters, signal was nearly independent of i.d. and better ion utilization was achieved at lower flow rates. Furthermore, emitters with a 50 µm i.d. and an etched o.d. produced about 1.5 times more signal than etched emitters with smaller i.d.s and about 3.5 times more signal than emitters with tapered inner and outer dimensions. Additionally, the work presented here has important implications for applications in which maximizing signal intensity and reducing frictional resistance to flow are necessary. Overall, the work provides an initial assessment of the critical parameters that contribute to maximizing the signal for electrospray ionization sources interfaced with pressure driven flows.

Analyte transport past a nanofluidic intermediate electrode junction in a microfluidic device.

A glass microfluidic device is presented in which a microchannel is split into two regions with different electric fields by a nanochannel intermediate electrode junction formed by dielectric breakdown. The objective is to sink current through the nanochannel junction without sample loss or broadening of the band as it passes the junction. This type of performance is desired in many microfluidic applications, including the coupling of microchannel/CE with ESI-MS, electrochemical detection, and electric field gradient focusing. The voltage offsets in this study are suitable for microchannel/CE-ESI-MS. Imaging of the transport of model anions and cations through the junction indicates that the junction exhibits nanofluidic behavior and the mean depth of the nanochannel is estimated to be approximately 105 nm. The ion permselectivity of the nanochannel induces concentration polarization and enriched and depleted concentration polarization zones form on opposite sides of the nanochannel, altering the current and electric field distributions along the main microchannel. Anion transport efficiency past the junction was high, 96.0%, and varied little over the pH range of 4.0-8.0. In contrast, cation transport is much lower, and decreases from 72 to 11% from pH 4.0 to 8.0. Band broadening increases with increasing pH less than 70% over the pH range of 4.0-8.0. It is anticipated that this characterization will aid in the understanding and optimization of such junctions made from permselective membranes and porous glass.

Simultaneous separation and detection of cations and anions on a microfluidic device with suppressed electroosmotic flow and a single injection point.

A rapid and simultaneous separation of cationic and anionic peptides and proteins in a glass microfluidic device that has been covalently modified with a neutral poly(ethylene glycol) (PEG) coating to minimize protein adsorption is presented. The features of the device allow samples that contain both anions and cations to be introduced from a central flow stream and separated in different channels with different outlets-all in the presence of low electroosmotic flow (EOF) imparted by the PEG coating. The analytes are electrophoretically extracted from a central hydrodynamic stream and electrophoretically separated in two different channels, in which pressure driven flow has been suppressed through the use of hydrodynamic restrictors. Having different outlets for the electrophoretic separation channels that are spatially separated from the injection enables coupling with further downstream functionalities or off-chip detection, such as mass spectrometry. A plug of charged analyte is hydrodynamically pumped to the sampling intersection and anions from the plug migrate electrophoretically toward the anode in one channel while cations migrate toward the cathode in the other channel due to suppressed EOF from the PEG coating. The separations presented here required less than a minute to complete and produced average separation efficiencies of up to about 3,500 plates from a separation length of 2 cm. The extraction efficiency of both cations and anions from the hydrodynamic stream is determined experimentally and compared with a previously reported model that was used to determine anion extraction efficiency. The extraction efficiency is determined to be 87% and 98% for the two sample mixtures analyzed, and the values predicted by the model are within 3.5% of the experimental data. It is anticipated that this basic approach for simultaneous separation of anions and cations with reduced EOF will be integrated into larger microfluidic systems because the design provides separate outlets that can feed downstream processes or linked to off-chip detection.

A theoretical and experimental study of the electrophoretic extraction of ions from a pressure driven flow in a microfluidic device.

The electrophoretic extraction of ions from a hydrodynamic flow stream is investigated at an intersection between two microfluidic channels. A pressure gradient is used to drive samples through the main channel, while ions are electrophoretically extracted into the side channels. Hydrodynamic restrictors and a neutral coating are used to suppress bulk flow through the side channels. A theoretical model that assumes Poiseuille flow in the main channel and neglects molecular diffusion is used to calculate the extraction efficiency, eta, as a function of the ratio, R, of the average hydrodynamic velocity to the electrophoretic velocity. The model predicts complete extraction of ions (eta=1) for R<2/3 and a monotonic decrease in eta as R becomes greater than 2/3, which agrees well with the experimental results. Additionally, the model predicts that the aspect ratio of the microfluidic channel has little effect on the extraction efficiency. It is anticipated that this device can be used for on-line process monitoring, sample injection, and 2D separations for proteomics and other fields.