Fast Surface Acoustic Wave-Matrix-Assisted Laser Desorption

Feb 5, 2013 - Fast Surface Acoustic Wave-Matrix-Assisted Laser Desorption Ionization Mass Spectrometry of Cell Response from Islets of Langerhans. Lor...
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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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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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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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Figure 8. Single islet MALDI spectrum obtained by stimulating (100 µM acetylcholine, 30 mM

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

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CONCLUSIONS

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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than 0.5 µL) of biofluid.

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FIGURES

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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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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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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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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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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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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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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

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in 5 to 1 ratio with 5 µM transferrin.

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

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

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Corresponding Author

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Staffan Nilsson.

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

392

Present Addresses

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

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55, DK-5230 Odense, Denmark

395

Author Contributions

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

397

to the final version of the manuscript.

398

Notes

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

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Analysdagarna (Sweden, '2012).

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ACKNOWLEDGMENT

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

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

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

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

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

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

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

411

Research Council.

412 413

ABBREVIATIONS

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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;

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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

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