Fast Surface Acoustic Wave-Matrix-Assisted Laser Desorption

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Fast SAW-MALDI MS of Cell Response from Islets of Langerhans Loreta Bllaci, Sven Kjellstrom, Lena Eliasson, James R. Friend, Leslie Y. Yeo, and Staffan Nilsson Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac3019125 • Publication Date (Web): 05 Feb 2013 Downloaded from http://pubs.acs.org on February 20, 2013

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Analytical Chemistry

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Fast SAW-MALDI MS of Cell Response from Islets

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of Langerhans

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Loreta Bllaci†, Sven Kjellström‡, Lena Eliasson ¥, James R. Friend§, Leslie Y. Yeo§, Staffan

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Nilsson†*

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†Lund University, Center for Chemistry and Chemical Engineering, Pure and Applied

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Biochemistry, Box 124, 221 00 Lund, Sweden

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‡Lund University, Center for Molecular Protein Science, Biochemistry and Structural Biology,

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Box 124, 221 00 Lund, Sweden.

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¥ Lund University, Diabetes Centre, Clinical Sciences Malmö, CRC 91-11, SE-205 02 Malmö,

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Sweden.

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§ RMIT University, Micro/Nanophysics Research Laboratory, School of Electrical & Computer

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Engineering, Melbourne, VIC, 3000Australia.

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KEYWORDS

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Open chip, Surface Acoustic Wave atomizer, MALDI MS, Rapid cell releasate profiling

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ACS Paragon Plus Environment

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ABSTRACT

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A desire for higher speed and performance in molecular profiling analysis at a reduced cost is

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driving a trend in miniaturization and simplification of procedures. Here we report the use of a

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Surface Acoustic Wave (SAW) atomizer for fast sample handling in MALDI MS peptide and

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protein profiling of Islets of Langerhans, for future Type 2 Diabetes (T2D) studies. Here the

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SAW atomizer was used for ultrasound (acoustic) extraction of insulin and other peptide

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hormones released from freshly prepared islets, stimulated directly on a membrane. A high

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energy propagating SAW atomizes the membrane-bound liquid into approximately 2µm

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diameter droplets, rich in cell-released molecules. Besides acting as a sample carrier, the

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membrane provides a purification step by entrapping cell clusters and other impurities within its

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fibers. A new SAW-based sample-matrix deposition method for MALDI MS was developed,

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characterized by a strong insulin signal and a limit of detection (LOD) lower than 100 attomoles,

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was achieved. Our results support previous work reporting the SAW atomizer as a fast and

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inexpensive tool for ultrasound, membrane-based sample extraction.. When interfaced with

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MALDI MS, the SAW-atomizer constitutes a valuable tool for rapid cell studies. Other

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biomedical applications of SAW-MALDI MS are currently being developed, aiming at fast

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profiling of biofluids. The membrane sampling is a simplistic and noninvasive collection method

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of limited volume biofluids such as the gingival fluid and the tearfilm.

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INTRODUCTION

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Over the years, bio-analysis has continually benefited at various levels from miniaturization,

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smaller sampling volumes1 and easier handling to dramatically shorten processing time. MALDI

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MS, an indispensable tool in protein analysis,2-4 is usually preceded by multiple steps of sample


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pretreatment and purification which not only are time consuming but might also lead to MALDI

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artifacts.5 Here we introduce the Surface Acoustic Wave (SAW) atomizer, a tool for fast

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ultrasound e.g. “mechanical” sample extraction6 from living cell samples in MALDI MS

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analysis.

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The atomizer consists of a low-loss piezoelectric substrate where the sample droplet is placed

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(Figure 1A) with two single-phase unidirectional transducers SPUDTs electrodes (Figure 1B)

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driven at 30 MHz in this study by an electrical supply.6-8 When the Rayleigh SAW propagates

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under the membrane-bound sample, SAW energy is transferred into the membrane (Figure 1C)

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and capillary waves7 are generated in the fluid on the sample's surface. Due to the tremendous

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accelerations induced by the 30 MHz frequency SAW atomizer, the capillary waves destabilize

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and ejects droplets from its crests; the aerosol generated possesses a nearly monodisperse

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diameter distribution centered at a value in the range of 2µm that depends on the fluid’s viscosity

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and surface tension.7 Cavitation is absent due to the fact that the propensity of cavitation

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nucleation is inversely dependent on the frequency, squared6-8 and at the frequencies used in the

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SAW, the power required is at least three orders of magnitude greater than used for atomization.

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The effect of fluid shear is minimized due to the short exposure time of only a few microseconds

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and the fact that the time scale of SAW-driven oscillation is far away from the typical resonant

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time scales of molecules that might incur shear-induced damage. An insignificant degradation of

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less than 1% was reported for SAW atomizer-extracted Ovalbumin and BSA for SDS-PAGE

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analysis.6 The open format of the SAW-atomizer,9 circumvents the use of sample transferring

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micro-channels,10, 11 and related clogging risk. Finally, SAW atomization is simple; is essentially

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instant-on, instant-off technology; economical with power consumption of less than 3W; and

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requires minimal personnel training. It is but one example of the burgeoning field of fluid and


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particle manipulation using acoustics that is solving a broad array of problems in microfluidics.6,

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Figure 1. The SAW atomizer’s working principle and experimental setup. The atomizer consists

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of a piezoelectric material with two SPUDT electrodes which generate unidirectional Rayleigh-

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wave SAW that propagate towards and underneath the sample. (A) A resting droplet on the SAW

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atomizer, on which no SAW excitation is present. (B) The SAW has reached the droplet,

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destabilized and generated liquid aerosol with a centered monodisperse diameter of 2 µm. (C)

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High energy SAW drive atomization even when the liquid is previously sampled on a membrane.

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The resulting aerosol (B and C) are collected on a MALDI plate via a pinhole for MS analysis.

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(D) Frontal image of the SAW atomizer (above); lateral image of the working atomizer (below).


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In combination with simple filter paper or with a purpose-selected membrane, upon application

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of the living cell sample, the SAW atomizer offers a purification and extraction system;

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impurities such as cell debris or cell clusters are likely to get entrapped within the membrane

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fibers. Successful SAW membrane-extraction of low molecular weight analytes from blood and

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water have already been demonstrated by interfacing SAW with ESI-MS.8,

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better suited for direct analysis of complex biological samples due to the relatively high tolerance

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of impurities and simplified spectra mostly consisting of single protonated species.13

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Nevertheless, multiple experimental factors determine the spectral quality, of which, the sample

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and matrix deposition on the MALDI plate is essential.14, 15 Ideally, the sample-matrix layer for

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analysis contains small, homogeneous and equally distributed matrix crystals embedding the

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sample analytes. Several deposition methods to date suffer from uneven analyte distribution

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within the MALDI spot,16 therefore manually searching for "hot” spots—regions of the

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preparation that yield higher signal intensities—is required. Thin matrix-sample layers are

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reported to ionize easily and lead to high signal intensities.17-19 The SAW-membrane platform is

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here used in fast screening of islet hormone secretion for future Type 2 Diabetes (T2D) studies in

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parallel to its use as a tool for thin layer MALDI sample preparation.

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MALDI MS is

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The disease arises from the interplay of several factors of which imbalance of pancreatic

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hormones, responsible for carbohydrate, fat, and protein metabolism regulation is a major

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contributor. Pancreatic hormones are produced from cell clusters named islets of Langerhans

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(Figure 2). Every islet harbors 1.000-3.000 cells of five different cell types named α, β, δ, PPi,

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and ε-cells. In human, insulin-releasing β-cells constitute the main population (48-59%) followed

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by the glucagon-releasing α-cells (33-46%).20, 21 Glucose is the main regulator of both insulin

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and glucagon release, while hormones22-24 or neurotransmitters like acetylcholine25 modulate the


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response from the islet’s cells. While impaired insulin release is an important factor for the

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disease onset, T2D patients are characterized by elevated levels of circulating glucagon,22

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suggesting that α-cells might also play a key role in the T2D pathophysiology. The contribution

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of other cell types—(δ, PPi, and ε-cells) constituting a minority of the islet mass—in the disease

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onset is even more obscure and requires further research.

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Figure 2. Illustration of an islet of Langerhans in mice. The β-cells (A), depicted in green,

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dominate the core of the islet, mostly surrounded by glucagon releasing α-cells (red). The

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electron microscopy (EM) picture (B) shows one insulin-releasing β-cell. Higher magnification

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of two adjacent β-cells shows numerous insulin-granules anchored in the plasma membrane. N-

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nucleus; g-granule; PM-plasma membrane; m-mitochondria. The green line indicates the plasma

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membrane. Scale bar 2 µm (B) and 0.5 µm (C).

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Comparative proteomics of healthy versus diseased islets as endocrine units might help

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identify T2D biomarkers and highlight T2D’s pathogenesis. In our forthcoming studies, we

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intend to use the SAW atomizer as a tool for quick sample handling in MALDI MS in this work.

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Besides performing an instant ultrasound-based, membrane purification/extraction of the sample,

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the chip reduces the use of vials, containers, and related analysis cost.


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EXPERIMENTAL SECTION

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Reagents. Acetylcholine chloride, ammonium citrate and bovine transferrin were purchased

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from Sigma-Aldrich (Buchs SG, Switzerland), human insulin from Novo Nordisk, α-cyano-4-

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hydroxycinnamic acid (CHCA) from Bruker Daltonics. TFA was purchased from Merck. Islets

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were freshly prepared and supplied from the Clinical Research Center (CRC), Malmö.

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Quantitative, medium-wide pores (5-6 µm pore size) filter papers, were provided by Munktell

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Filter AB (Falun, Sweden). Acetonitrile and MilliQ water were of LC gradient grade. RPMI-

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1640 (Roswell Park Memorial Institute medium) media was purchased from Sigma Aldrich

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while

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phosphoethanolamine, triethylammoniumsalt) from Invitrogen UK.

Fluorescein-DHPE(N-(fluorescein-5-thiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-

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Biological Sample Preparation. Mice used in the experiments were female NMRI

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(Crl:NMRI(Han) (outbred), 8 weeks old. Approximately 100 islets were isolated from each

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mouse, allowing for 15 to 20 performed experiments. After isolation, islets were suspended in

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RPMI 1640 (Roswell Park Memorial Institute medium). Prior to the analysis they were

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transferred in HEPES-buffered medium, pH 7,4: 25 mM HEPES, 125 mM NaCl, 5.9 mM KCl,

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12.8 mM CaCl2, 1.2 mM MgCl2, with various concentrations of stimulating glucose content

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varying from 10 to 30 mM. For the control experiments, the buffer was supplied with 3 mM

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glucose; a concentration below the insulin-release threshold (7 mM),26 and acetylcholine varying

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in concentration (0, 1 and 100 µM). Acetylcholine chloride for stimulation of islets and bovine

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transferrin (used as a matrix additive in the insulin LOD evaluation) were prepared at

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concentrations of 100, 10 and 5 µM respectively.

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Stimulation of islets on the atomizer. A volume of 2 µL HEPES buffer, containing typically

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2-6 islets, was pipetted on a dry, un-pretreated membrane placed on the atomizer. An equivalent


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volume of acetylcholine (aq) was subsequently applied on the islets located onto the membrane.

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Although not being the main grounds of this work, stimulation of a single islet was performed on

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the bare chip without employing the membrane. In this case, 1 µL HEPES buffer containing one

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islet was placed on the chip followed by the addition of 1 µL acetylcholine (aq). Stimulation time

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with acetylcholine was five minutes in all experiments. Stimulated islets, either on the membrane

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or directly on the chip, were supplied every 2 minutes with 0.5µL water to compensate for the

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evaporation.

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Electron Micrscopy. The samples in the electron micrographs were fixed in 2.5%

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glutaraldehyde and 1% osmiumtetroxide prior to embedding in AGAR100 as previously

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described by Andersson et al.27 Images were taken using a JEM 1230 electron microscope.

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Fluorescent Nanoparticles. Lipid-based nanoparticles were prepared as described by Nilsson28 with supplementary fluorescein DHPE.

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Surface Acoustic Wave atomizer. The SAW atomizer consists of a low-loss piezoelectric,

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single crystal lithium niobate 127.68° Y-rotated cut, X-propagating material sputtered with two

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chromium–aluminum single-phase unidirectional transducer (SPUDT). When a sinusoidal

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electrical signal is applied between the electrodes, Rayleigh surface acoustic waves of a few nm

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in amplitude are produced and propagate unidirectionally along the X-axis of the substrate,

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toward and underneath the membrane-bound sample. The transverse component of the SAW is

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progressively absorbed into the fluid, generating a simple sound wave that propagates at the so-

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called Rayleigh angle that is only dependent upon the relative difference in sound speed between

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the SAW in the lithium niobate and the sound wave in the fluid.8 The sound wave propagating in

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the fluid drives the formation of a capillary wave along the fluid present atop the membrane, and

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due to the extreme accelerations of over 108 m/s2 present at the fluid interface, droplets are


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driven from the crests of the destabilized capillary wave without cavitation to form a micron-

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order aerosol.6 The SAW-atomizer was fabricated in house by the Melbourne Centre for

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Nanofabrication.

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Matrix preparation. In our experiments, 2-5 mg/mL CHCA was dissolved in 50: 50: 0,1 %

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with water, acetonitrile and trifluoroacetic acid (TFA) with supplementary 2mM ammonium

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citrate. In the LOD evaluation experiments, Transferrin (5 µM and 10 µM ) was used as a matrix

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additive29 for insulin signal enhancement (Figure 7, pink trace).

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Stainless steel plate mask. A plate mask, set atop a plastic surface possessing a 10 mm

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diameter opening corresponding to 10 neighbor spots on the MALDI plate, was used to prevent

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the aerosol’s deposition throughout the plate's area. The membrane size was 5x 8 mm.

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MALDI MS Instrument. MALDI-MS analyses of the crystallized samples were performed

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on a 4700 Proteomics Analyzer MALDI-TOF/TOF™ mass spectrometer (Applied Biosystems,

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Framingham, MA). MS data acquisition (3000 laser shots per spot) was performed in positive

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linear mode. Stainless steel plates with 192 positions were used for the trapping of SAW-

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generated aerosol.

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Sample-matrix deposition method. Sample and matrix were consecutively SAW-extracted

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from the membrane and deposited onto a controlled area of the MALDI plate. Every SAW

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extraction/deposition event takes less than 15 seconds, therefore sample and matrix SAW

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extraction/deposition takes approximately 30 seconds. The sample was prepared as previously

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described (see, “Stimulation of islets on the atomizer”). A volume of 4 µL CHCA was used for

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matrix-membrane saturation, following the first event of SAW extraction/deposition of the

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sample. When a single islet was stimulated on the chip (no membrane used), a volume of 2 µL

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CHCA was applied on the cleaned atomizer.


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SAW atomizer cleaning. After each experiment, the piezoelectric substrate was cleaned with acetone, isopropanol and rinsed with milliQ water as described by J. Ho.8

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RESULTS AND DISCUSSION

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Fluorescent microscopy visualization of SAW extracted fluorescent nanoparticles

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First, a simple approach to demonstrate the SAW operation was carried out through the

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extraction of fluorescent nanoparticles28. Nanoparticles extracted from the membrane via SAW

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atomization were collected on a second membrane that was subjected to conventional fluorescent

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microscopy (FM, Figure 3). The increases in nanoparticle concentration lead to enhanced FM

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signaling suggesting the role of the SAW in the extraction process.

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Figure 3. Visualization of SAW-extracted fluorescent nanoparticles atop a collection membrane.

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Nanoparticles embedded in a membrane placed on the atomizer are extracted via SAW

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atomization and collected on a second membrane positioned parallel to and above the atomizer,

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in a fashion similar to the MALDI plate. The collection membrane shows numerous SAW-

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extracted fluorescent nanoparticles; wherein the brighter spots represent aggregates of the

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extracted nanoparticles.

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SAW-MALDI MS spectra of healthy islet secretion

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Fast MALDI MS spectra of stimulated, intact islets from healthy mice are shown in Figure 4.

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Islet’s chemical stimulation (30 mM glucose, 100 µM acetylcholine) has selectively triggered the

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healthy β–cells to release insulin (Figure 4), therefore the acquired spectra are characterized by

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intense peaks of insulin (relative intensity). Other co-secreted molecules such as C-peptide and

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amylin are also detected. Acetylcholine can evoke insulin release under non-stimulatory glucose

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(3mM) at high concentrations (100µM) only. However, in this case, insulin release is

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considerably reduced (spectra not shown).

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The second largest cell community of the islet, α-cells, respond completely opposite to β-cells

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towards glucose concentrations. While high glucose above 7 mM triggers mouse β-cells to

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release insulin, glucagon-secretion at these higher concentrations is suppressed.30 Low levels of

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glucagon in healthy individuals keep the glycemic index in control by suppressing hepatic

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glucose output. However T2D is characterized by reduced glucagon suppression,22 a subject

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which has gained high interest in the recent years.

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Other α-cells released peptides like glicentin-related polypeptide (GRPP), the incretin

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glucagon-like 1 (GL-1), and δ-cell’s somatostatin-14 peptide (SMS-14) are also indicated in the

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spectra (Figure 4).


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Figure 4. SAW-MALDI MS spectra of stimulated islets of healthy mice. (A) Acquired spectra

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from three islets in 30 mM Glucose HEPES using the SAW-spotting. Single-charged insulin

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(5807 Da), glucagon (3482 Da), and SMS-14 (1640 Da) peptide are labeled. (B) A magnification

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of the preceding spectra reveals several peaks, corresponding to single-charged peptides released

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from α-cells: GRPP (3439 Da), GLP-1(4168 Da), β-cell released C-peptide (3121 Da), amylin

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(3922 Da), somatostatin (SMS-14, 1640 Da) released from δ-cells. The double charged insulin is

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also detected (2905 Da).

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Sample-matrix deposition method


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In MALDI MS, informative and qualitative spectra acquisition is greatly depended on the

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procedure how the sample and matrix is deposited on the MALDI plate.14, 31 Additives to the

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matrix solution are also important; it was shown previously that the addition of phosphoric acid32

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or transferrin29 to the matrix solution improves the detectability of intact protein. Our acquired

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spectra, characterized by high signal intensities and “run to run” reproducibility have been

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acquired with the “SAW-sampling/SAW-matrixing” on the plate.

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According to our developed method, the membrane-bound sample is SAW-extracted and

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deposited on the MALDI plate (Figure 5). In a second step the same volume of matrix is applied

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on the membrane, subsequently, SAW-extracted and deposited atop the sample film on the

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MALDI plate. The overall time of SAW sample/matrix deposition takes approximately 30

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seconds, where every SAW extraction event takes less than 15 seconds.

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Figure 5. Illustration of the SAW-deposition method. (A) The membrane-bound sample is first

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extracted with SAW atomization and deposited on a controlled area (by a pinhole in the plastic


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mask) of the MALDI plate. (B) Following sample extraction, the matrix solution is applied on

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the same membrane and subsequently deposited on the plate using the SAW. (C) Depiction of

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the area where the thin film of SAW-extracted sample and matrix are deposited on the MALDI

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plate.

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The SAW-deposition method resulted in thin layer formation of the sample and matrix that

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ionizes easily and is characterized by high signal intensities.17-19 Spraying sample and matrix on

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the MALDI plate with ad Electrospray needle and piezoelectric microdispenser33 has also been

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reported to form homogenous matrix-sample crystals characterized by high signal enhancement

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and reproducibility.34-37 Besides its obvious advantages, electrospraying the matrix-sample

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mixture nevertheless has its downsides, including the necessity of extra equipment (e.g.,

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transformers, etc.) and dangerously high voltages (3-5 kV). Thus, another advantage of the SAW

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chip emerges in our study as an easy, fast and safe tool for thin matrix-sample crystal layer

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deposition for MALDI MS.

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Initially we tested the “Quick & Dirty”, the “Matrix-precoated layers” and the “Fast

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evaporation” methods of sample/matrix deposition.15 In any method, the matrix: analyte volume

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ratio38 (under matrix molar excess conditions) is of particular importance. However small matrix

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pipetted volumes (0.5 µL) inundated the thin film of the SAW-extracted sample leading to loss

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of signal.

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Reproducibility and LOD

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The SAW-deposition strategy favors thin sample layer formation, associated with high signal

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intensities and reproducibility (Figure 6). The acquired MALDI spectra of stimulated intact islets

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are characterized by high S/N values of single charged insulin.


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Figure 6. The MALDI MS spectra acquired and shown in this figure, were acquired by

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stimulation of four Spectra acquired and shown in this figure, were acquired by stimulation of

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four islets. Each MALDI spectra is characterized by a large peak corresponding to the single-

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charged mouse insulin (MW 5807 Da) and a small peak of single charged glucagon (MW 3482

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Da). The double-charged insulin (2905 Da) is also detected. In the inset a magnified view of the

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islet’s response spectra is shown. Thorough investigation of low abundance peptide and proteins

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is needed to find new T2D markers resulting in a better understanding of the molecular

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mechanisms underlying the T2D disease.

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The lower insulin LOD, estimated by serial dilutions of insulin stock solutions, using the

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SAW-deposition method was less than 100 attomole, with 2 mg/mL CHCA as a matrix (Figure

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7). The signal was significantly enhanced, by at least one order of magnitude, when the matrix

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was premixed with 5 µM transferrin in a 5 to 1 ratio. Our results are in good agreement with


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previous studies suggesting enhanced properties of transferrin when premixed with matrices.29

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The enhancing properties of transferrin-premixed matrices on insulin and other protein/peptides

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released by stimulated islets will require further study. 300 301 302 303 304 305 306 307

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Figure 7. MALDI spectra obtained from SAW-spotting of 100 attomole human insulin (MW

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5808 Da) without (blue trace) and with transferrin (red trace) in the matrix (2 mg/mL) premixed

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in 5 to 1 ratio with 5 µM transferrin.

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Islet response sampled on the bare chip

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The membrane, as mentioned earlier, aids sample pre-purification, “desalting” and thin crystal

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layer formation on the MALDI plate leading to qualitative spectra. However the atomization

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could be carried out without the membrane when "pure sample” is used and thus decreasing the

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risk of membrane sample retention that could be critical for detecting scarce analytes. Figure 8

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shows the MALDI spectra from a freshly prepared and thoroughly washed single islet stimulated

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directly on the bare chip. The acquired spectra are characterized by a good baseline; signals

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corresponding to insulin, C-peptide, glucagon and other small peptides, are also detected.


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321 322

Figure 8. Single islet MALDI spectrum obtained by stimulating (100 µM acetylcholine, 30 mM

323

Glucose, HEPES buffer) the islet, present on the naked chip, e.g. without membrane purification.

324 325

CONCLUSIONS

326

The SAW atomizer can be used as a fast, ultrasound extraction tool for small volume, complex

327

samples from a membrane. We have successfully for the first time (to our knowledge)

328

hyphenated the SAW open chip with MALDI MS. In our experiments it was used for the

329

extraction of islet hormones released upon stimulation with glucose and acetylcholine for future

330

T2D studies. Since the sample here is minimally chemically pretreated, the risk of MALDI

331

artefacts is considerably reduced. Furthermore, we have developed a SAW-based sample-matrix

332

deposition method that generates MALDI spectra characterized by high signal intensities of the

333

analytes with high “run to run” reproducibility. The LOD for insulin was 100 attomole, however

334

when transferrin was premixed with the matrix the signal was enhanced to 10 attomoles.

335

Reduced sample handling analysis times, qualitative MALDI spectra acquisition, and membrane

336

sample preservation reduces the overall cost of biomolecular screening such as comparative MS

337

protein profiling for T2D biomarkers. Future use of SAW-MALDI MS in single cell studies can


17


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338

be foreseen. We are currently investigating other applications of SAW-MALDI, such as relative

339

quantification and rapid peptide and protein profiling of membrane preserved, small volume (less

340

than 0.5 µL) of biofluid.

341 342

FIGURES

343

Figure 1. The SAW atomizer’s working principle and experimental setup. The atomizer consists

344

of a piezoelectric material with two SPUDT electrodes which generate unidirectional Rayleigh-

345

wave SAW that propagate towards and underneath the sample. (A) A resting droplet on the SAW

346

atomizer, on which no SAW excitation is present. (B) The SAW has reached the droplet,

347

destabilized and generated liquid aerosol with a centered monodisperse diameter of 2 µm. (C)

348

High energy SAW drive atomization even when the liquid is previously sampled on a membrane.

349

The resulting aerosol (B and C) are collected on a MALDI plate via a pinhole for MS analysis.

350

(D) Frontal image of the SAW atomizer (above); lateral image of the working atomizer (below).

351

Figure 2. Illustration of an islet of Langerhans in mice. The β-cells (A), depicted in green,

352

dominate the core of the islet, mostly surrounded by glucagon releasing α-cells (red). The

353

electron microscopy (EM) picture (B) shows one insulin-releasing β-cell. Higher magnification

354

of two adjacent β-cells shows numerous insulin-granules anchored in the plasma membrane. N-

355

nucleus; g-granule; PM-plasma membrane; m-mitochondria. The green line indicates the plasma

356

membrane. Scale bar 2 µm (B) and 0.5 µm (C).

357

Figure 3. Visualization of SAW-extracted fluorescent nanoparticles atop a collection membrane.

358

Nanoparticles embedded in a membrane placed on the atomizer are extracted via SAW

359

atomization and collected on a second membrane positioned parallel to and above the atomizer,


18

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360

in a fashion similar to the MALDI plate. The collection membrane shows numerous SAW-

361

extracted fluorescent nanoparticles; wherein the brighter spots represent aggregates of the

362

extracted nanoparticles.

363

Figure 4. SAW-MALDI MS spectra of stimulated islets of healthy mice. (A) Acquired spectra

364

from three islets in 30 mM Glucose HEPES using the SAW-spotting. Single-charged insulin

365

(5807 Da), glucagon (3482 Da), and SMS-14 (1640 Da) peptide are labeled. (B) A magnification

366

of the preceding spectra reveals several peaks, corresponding to single-charged peptides released

367

from α-cells: GRPP (3439 Da), GLP-1(4168 Da), β-cell released C-peptide (3121 Da), amylin

368

(3922 Da), somatostatin (SMS-14, 1640 Da) released from δ-cells. The double charged insulin is

369

also detected (2905 Da).

370

Figure 5. Illustration of the SAW-deposition method. (A) The membrane-bound sample is first

371

extracted with SAW atomization and deposited on a controlled area (by a pinhole in the plastic

372

mask) of the MALDI plate. (B) Following sample extraction, the matrix solution is applied on

373

the same membrane and subsequently deposited on the plate using the SAW. (C) Depiction of

374

the area where the thin film of SAW-extracted sample and matrix are deposited on the MALDI

375

plate.

376

Figure 6. The MALDI MS spectra acquired and shown in this figure, were acquired by

377

stimulation of four Spectra acquired and shown in this figure, were acquired by stimulation of

378

four islets. Each MALDI spectra is characterized by a large peak corresponding to the single-

379

charged mouse insulin (MW 5807 Da) and a small peak of single charged glucagon (MW 3482

380

Da). The double-charged insulin (2905 Da) is also detected. In the inset a magnified view of the

381

islet’s response spectra is shown. Thorough investigation of low abundance peptide and proteins


19


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Page 20 of 24

382

is needed to find new T2D markers resulting in a better understanding of the molecular

383

mechanisms underlying the T2D disease.

384

Figure 7. MALDI spectra obtained from SAW-spotting of 100 attomole human insulin (MW

385

5808 Da) without (blue trace) and with transferrin (red trace) in the matrix (2 mg/mL) premixed

386

in 5 to 1 ratio with 5 µM transferrin.

387

Figure 8. Single islet MALDI spectrum obtained by stimulating (100 µM acetylcholine, 30 mM

388

Glucose, HEPES buffer) the islet, present on the naked chip, e.g. without membrane purification.

389

Corresponding Author

390

Staffan Nilsson.

391

E-mail: [email protected]; Phone +46 46 222 81 77; fax:+46 46 222 46 11

392

Present Addresses

393

†Institute of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej

394

55, DK-5230 Odense, Denmark

395

Author Contributions

396

The manuscript was written through contributions of all authors. All authors have given approval

397

to the final version of the manuscript.

398

Notes

399

Restricted parts of this work have been presented in MS-Öresund (Sweden, 2011) and

400

Analysdagarna (Sweden, '2012).

401


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402 403

ACKNOWLEDGMENT

404

The Swedish Research Council (VR no 621-2010-5384 to S. Nilsson). We owe our gratitude to

405

Britt-Marie S Nilsson for islets preparation. LYY and JRF acknowledge support of the SAW

406

atomizer technology by the Australian Research Council (DP0985253, DP1092955, and

407

DP120100013) and the NHMRC for Development Grant 1000513. JRF is grateful to the

408

Melbourne Centre for Nanofabrication for an MCN Tech Fellowship and RMIT University for

409

the Vice-Chancellor’s Senior Research Fellowship. LYY is grateful for an Australian Research

410

Fellowship from the Australian Research Council. LE is a senior researcher at the Swedish

411

Research Council.

412 413

ABBREVIATIONS

414

SAW, surface acoustic wave;T2D, type 2 diabetes; LOD, limit of detection; SPUDT, single-

415

phase unidirectional transducer; CHCA, α-cyano-4-hydroxycinnamic acid; SMS, somatostatin;

416

GLP-1, glucagon-like peptide 1; SMS-14, somatostatin-14; GRPP, glicentin-related polypeptide.

417

Fluorescein-DHPE(N-(fluorescein-5-thiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-

418

phosphoethanolamine, triethylammoniumsalt) RPMI 1640 (Roswell Park Memorial Institute

419

medium).

420 421

REFERENCES


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

422 423

Page 22 of 24

(1) Santesson, S.; Degerman, E.; Rorsman, P.; Johansson, T.; Lemos, S.; Nilsson, S. Integr. Biol. 2009, 1, 595-601.

424

(2) Karas, M.; Bachmann, D.; Hillenkamp, F. Anal. Chem. 1985, 57, 2935-2939.

425

(3) Karas, M.; Bachmann, D.; Bahr, U.; Hillenkamp, F. Int. J. Mass Spectrom. Ion Processes

426

1987, 78, 53–68.

427

(4) Karas, M; Hillenkamp, F. Anal. Chem. 1988, 60, 2299–2301.

428

(5) Walch, A.; Rauser, S.; Deininger, S. O.; Höfler, H. Histochem. Cell Biol. 2008, 130, 421–

429

434.

430

(6) Qi, A.; Yeo, L. Y.; Friend, J. R.; Ho, J. Lab Chip 2010, 10, 470-476.

431

(7) Qi, A.; Yeo, L. Y.; Friend, J. R. Phys. Fluids 2008, 20, 074103.

432

(8) Ho, J. T.; M. K.; Go, D. B.; Yeo, L. Y.; Friend, J. R.; Chang, H. C. Anal. Chem. 2011, 83,

433

3260–3266.

434

(9) Yeo, L. Y.; Friend, J. R. Biomicrofluidics 2009, 3, 012002.

435

(10) Dishinger, J. F.; Reid, K. R.; Kennedy, R. T. Anal. Chem. 2009, 81, 3119-3127.

436

(11) Lenshof, A.; Magnusson, C.; Laurell, T. Lab Chip 2012, 12, 1210-1233.

437

(12) Heron, S. R.; Wilson, R.; Shaffer, S. A.; Goodlett, D. R.; Cooper, J. M. Anal. Chem. 2010,

438 439 440

82, 3985-3989. (13) Hummon, A. B.; Sweedler, J. V.; Corbin, R. C. TrAC, Trends Anal. Chem. 2003, 22, 515– 521.

441

(14) Cohen, S. L.; Chait, B. T. Anal. Chem. 1996, 68, 31-37.

442

(15) Sigma-Aldrich. AnalytiX 2001, 6, 1-6.

443

(16) Garden, R. W.; Sweedler, J. V. Anal. Chem. 2000, 72, 30-36.

444

(17) Knochenmuss, R. Anal. Chem. 2003, 75, 2199-2207.


22

Page 23 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


445

(18) Knochenmuss, R.; Zhigilei, L. V. J. Phys. Chem. 2005, 109, 22947-22957.

446

(19) Knochenmuss, R. Analyst (Cambridge, U. K.) 2006, 131, 966-986.

447

(20) Cabrera, O.; Berman, D. M.; Kenyon, N. S.; Ricordi, C.; Berggren, P.O.; Caicedo, A.

448 449 450 451 452 453 454 455 456 457 458

PNAS 2006, 103, 2334 –2339. (21) Brissova, M.; Fowler, M. J.; Nicholson, W. E.; Chu, A.; Hirshberg, B.; Harlan, D.; Power, A. C. J. Histochem. Cytochem. 2005, 53, 1087-1097. (22) Lund, A.; Vilsbøll, T.; Bagger, J. I.; Holst, J. J.; Knop, F. Am. J. Physiol. Endocrinol. Metab. 2011, 300, 1038-1046. (23) Hansen, K. B.; Vilsbøll, T.; Bagger, J. I.; Holst, J. J.; Knop, F. K. J. Clin. Endocrinol. Metab. 2012, 97, 1363-1370. (24) Knop, F. K.; Aaboe, K.; Vilsbøll, T.; Vølund, A.; Holst, J.; Krarup,T.; Madsbad, S. Obes. Metab. 2012, 14, 500-510. (25) Gromada, J.; Høy, M.; Renström, E.; Bokvist, K.; Eliasson, L.; Göpel, S.; Rorsman, P. J. Physiol. 1999, 518, 745-759.

459

(26) Gilon, P.; Henquin, J. C. Endocr. Rev. 2001, 22, 565–604.

460

(27) Andersson, S. A.; Pedersen, G.; Vikman, J.; Eliasson, L. Pflugers Arch. - Eur. J. Physiol.

461 462 463 464 465 466 467

2011, 462, 443-454. (28) Nilsson, C.; Harwigsson, I.; Becker, K; Bülow, L.; Birnbaum, S.; Nilsson, S. Anal. Chem. 2009, 81, 315-321. (29) Kobayashi, T.; Kawai, H.; Suzuki, T.; Kawanishi, T.; Hayakawa, T. Rapid Commun. Mass Spectrom. 2004, 18, 1156-1160. (30) MacDonald, P. E.; De Marinis, Y. Zh.; Ramracheya, R.; Salehi, A.; Ma, X.; Johnson, P. R. V.; Cox, R.; Eliasson, L.; Rorsman, P. PLoS Biol. 2007, 5, 1236-1247.


23


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 24

468

(31) Beavis, R. C.; Chait, B. C. Methods Enzymol. 1996, 270, 519-551.

469

(32) Kjellström, S.; Jensen, O. N. Anal. Chem. 2004, 76, 5109-5117.

470

(33) Miliotis, T.; Kjellstrom, S.; Nilsson, J.; Laurell, T.; Edholm, L. E.; Marko-Varga, G. .

471 472 473 474 475

Rapid Commun. Mass Spectrom. 2002, 16, 117-126. (34) Axelsson, J.; Hoberg, A. M.; Waterson, C.; Myatt, P.; Chield, G. L.; Varney, J.; Haddleton, D. M.; Derrick, J. Rapid Commun. Mass Spectrom. 1997, 11, 209-213. (35) Hensel, R. R.; King, R. C.; Owens, K. G. Rapid Commun. Mass Spectrom. 1997, 11, 1785-1793.

476

(36) Hanton, S.; Clark, P.; Owens, K. J. Am. Soc. Mass Spectrom. 1999, 10, 104-111.

477

(37) Yao, J.; Scott, J. R.; Young, M. K.; Wilkins, C. L. J. Am. Soc. Mass Spectrom. 1998, 9,

478 479 480

805-813. (38) Duncan, M. W.; Roder, H.; Hunusucker, S. W. Brief. Funct. Genomic. Proteomic. 2008, 7, 355-370.

481 482


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Fast Surface Acoustic Wave-Matrix-Assisted Laser Desorption

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