Direct Biological Sample Analyses by Laserspray Ionization Miniature

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Direct Biological Sample Analyses by Laserspray Ionization Miniature Mass Spectrometry Yanbing Zhai, Siyu Liu, Lijuan Gao, Lili Hu, and Wei Xu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b05366 • Publication Date (Web): 21 Mar 2018 Downloaded from http://pubs.acs.org on March 21, 2018

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Analytical Chemistry

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Direct Biological Sample Analyses by Laserspray

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Ionization Miniature Mass Spectrometry

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Yanbing Zhai,1# Siyu Liu,1# Lijuan Gao,2 Lili Hu,1 and Wei Xu1*

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School of Life Science, Beijing Institute of Technology, Beijing 100081, China

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Beijing Center Physical and Chemical Analysis, Beijing, 100089, China

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*Corresponding Author: Wei Xu School of Life Science Beijing Institute of Technology Haidian, Beijing, 100081, China Email: [email protected] Website: http://www.escience.cn/people/weixu

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#Equal Contribution

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ACS Paragon Plus Environment

Analytical Chemistry 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

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Abstract

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With improved performances, miniature mass spectrometers are becoming

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suitable for more practical applications. At the same time, the coupling of an

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approximate ionization source is essential in terms of minimizing sample preparation

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and broadening the range of samples that could be analyzed. In this study, an

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atmospheric pressure laserspray ionization (AP-LSI) source was coupled with our

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home developed miniature ion trap mass spectrometer. The whole system is compact

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in size, and biological samples could be directly analyzed with minimum sample

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preparation. Direct detections of peptides, proteins, drugs in whole blood and urine

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could be achieved with high sensitivity. The analyses of tissue sections were

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demonstrated, and different regions in a tissue section could be differentiated based

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on their lipid profiles. Results suggest that the coupling of AP-LSI with miniature

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mass spectrometer is a powerful technique, which could potentially benefit target

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molecule analysis in biological and medical applications.

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1. Introduction

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Mass spectrometry (MS) is a powerful analytical technique, which has been

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widely applied in the analyses of chemical and biological molecules.1-5 Conventional

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laboratory MS instruments have high analytical performances in terms of sensitivity,

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mass range and mass resolution. However, these instruments are also typically

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large-in-size and high in power consumption. Miniature MS systems have then been

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developed to meet the increasing demands of on-site chemical analysis.6-8 Based on

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membrane inlet9-11 or ultra-low gas intake techniques,12,13 various portable gas

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chromatography MS (GC-MS) systems become available for volatile sample analysis

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in the field.14-16 To handle non-volatile samples, atmospheric pressure interfaced (API)

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miniature mass spectrometers were also developed.17-22 An atmospheric pressure

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interface enables the coupling of atmospheric pressure ionization sources with a

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miniature mass spectrometer, so that non-volatile samples such as biomolecules could

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be analyzed. At the same time, great efforts have also been made to improve the

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performances of a miniature mass spectrometer, especially in the past five years. For

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example, sensitivity of the continuous atmospheric pressure interfaced (CAPI)

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miniature mass spectrometer have been improved by ~ 100 times from ~1 µg/mL to

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~10 ng/mL when analyzing peptides.17,19,23

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With enhanced performances, miniature mass spectrometers are ready and have

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been used in more applications, such as from toxic gas and explosive detection to

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point-of-care testing.13,24-29 When using a miniature mass spectrometer, it is expected

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to be easy-to-operate and fast-in-analysis, which is essentially different from

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laboratory applications. In practical application scenarios, there are minimum sample

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pretreatment resources. Instruments, such as centrifugal and chemical dryer devices,

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will not be available. Therefore, a miniature mass spectrometer is normally facing

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samples in complex matrices directly, and there is a higher requirement in terms of

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handling complex matrix. The coupling of ambient or atmospheric pressure ionization

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methods30-32 with miniature mass spectrometer is a perfect marriage, which could

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effectively solve this problem. Indeed, several ambient ionization techniques have 3



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been coupled with miniature mass spectrometers and demonstrated powerful in

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different applications. Up to now, electrospray33-36 and plasma18,37-39 based ionization

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methods as well as matrix assisted ionization (MAI)40 have been coupled with

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portable mass spectrometers. Easy in sample preparation, powerful in analyte

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desorption and ionization, atmospheric pressure matrix assisted laser desorption

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ionization (AP-MALDI)41,42 method has also been coupled with a medium-size,

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field-deployable mass spectrometer.43,44 Even so, a conventional AP-MALDI source

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is not the best choice for miniature mass spectrometers. One reason is that the mass

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range of a miniature mass spectrometer is limited (typically < 2000 Th). A

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conventional AP-MALDI source typically generates ions with single charge, and

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mass spectrometers with higher mass ranges may be necessary. As another laser based

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ionization method, laserspray ionization (LSI)45,46 is a variant of MAI. In LSI, a laser

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is applied to desorb solid samples from a substrate followed by an ionization process,

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which is believed to be assisted by vacuum, matrix and heated capillary. LSI is easy

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to operate and even could be performed at a normal AP-MALDI or MALDI setup

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under their operating conditions.47,48 More importantly, LSI could produce highly

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charged gas-phase ions directly from solid samples,45,49,50 which is the significant

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difference from conventional MALDI. As a result, LSI would be favorable when

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coupling with miniature mass spectrometers.

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In this study, an IR pulsed laser was used for an atmospheric pressure LSI

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(AP-LSI) and coupled with our home-developed CAPI mini mass spectrometer. Prior

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to this study, IR lasers have been used in conventional MALDI and LSI but coupled

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with lab-scale instruments.49,51,52 Herein, the direct analyses of biological samples

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were demonstrated using this AP-LSI mini MS system. By choosing appropriate

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matrix and sample preparation method, this AP-LSI source could generate ions with

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multiple charges, especially for peptides and proteins. As a result, proteins, such as

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cytochrome C and myoglobin could be detected within a mass range below 1500 Th.

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When analyzing pure samples, tens of picogram detection sensitivity was achieved for

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drugs and peptides. This system shows high tolerance to complex matrices. Drugs in 4


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whole blood and urine could be detected with a limit of detection (LOD) on the level

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of couple hundreds of ng. The analysis of tissue samples was also demonstrated. High

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throughput MS analysis using this standalone fieldable mass spectrometer could be

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achieved by working with a moving stage and optimized matrices, which could

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potentially facilitate targeted biomarker detections in point-of-care testing.

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2. Experimental sections

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2.1 Instrumentation

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In this study, an AP-LSI source was coupled with our home developed miniature mass

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spectrometer, which has a continuous atmospheric pressure interface.17,19 As shown in

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Figure 1a, a pulsed diode-pumped solid-state (DPSS) laser was chosen to be used in

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the AP-LSI source. This DPSS laser source (Wedge 1064, Brightness Solutions, Italy)

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is relatively small-in-size, and it can output a pulsed infrared laser beam with a

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wavelength of 1064 nm and a pulse width of 1 ns. Its pulse frequency could be

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adjusted ranging from 1 Hz to 1000 Hz, and energy of each pulse is also adjustable up

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to 4 mJ per pulse energy. A series of optical lens were used to guide the laser beam to

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the sample holder placed right below the MS inlet. To ensure stability of the laser path,

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all optical components were integrated onto a steel plate. The laser beam was finally

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focused by a focusing lens with a focal length of 25 mm, and a laser spot size of ~0.6

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mm (in diameter) is used in this study. All optics lenses were purchased from Daheng

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Optics Inc. (Beijing, China). For safety consideration, protection goggles and a

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laboratory coat must be worn during experiments.

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The miniature mass spectrometer was developed in house, details about the

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instrument setup could be found in our previous publications.17-19 Briefly, the mini

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MS was based on a differential pumping system with two vacuum chambers, which

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were connected to each other with a skimmer, and a stainless steel capillary was used

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to the connect the first vacuum chamber with the atmosphere environment. This

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capillary also served as sample inlet and had an inner diameter of 0.25 mm and a

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length of 20 cm. As one type of inlet ionization method,40,45,53,54 the ionization process

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of LSI in this study occurred in the inlet capillary, and laser ablation helps in terms of

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analyte desorption. During experiments, the inlet capillary was grounded and exposed

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in room temperature. A linear ion trap with hyperbolic electrodes was placed in the

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second vacuum chamber and served as the mass analyzer. An electron multiplier

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(model 2500, Detech Inc.) integrated with a dynode was used as the ion detector.

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Established in this work, the whole instrument, including the laser source, was around 6


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60 × 45 × 26 cm in dimensions (38 × 26 × 24 cm when leaving out the laser source)

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and less than 20 kg in total weight. The system was pumped by the combination of a

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turbo pump (10 L/s, Hipace 10, Pfeiffer Inc., Germany) and a diaphragm pump (50

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L/min, SVF-E0-50, Scroll Tech Inc., China), which maintain an operational pressure

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of ~6 mTorr for the ion trap. In addition, the mass range of the instrument was

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adjusted up to around 2000 Th by lowering the RF frequency. Experiments were

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carried out in a repetitive laser shot mode with a pulse frequency of 10 Hz, unless

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otherwise specified. In this study, the laser pulse was not synchronized with the

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mini-MS. No voltage was applied to the sample plate. The optimized mini-MS

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operating parameters were found according to a standard tuning procedure using

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nano-ESI. The ion injection time was typically set to 500-1000 ms depending on the

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analyte amount or concentration. The experiments presented in this work were all

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performed in a positive ion mode.

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2.2 Chemical reagents and samples

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Chemical reagents. Reserpine (MW 608.68) was purchased from Acros

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Organics (Geel, Belgium), Ciprofloxacin (MW 331.34) was purchased from Aladdin

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industrial. Inc (Shanghai, China). High-performance liquid chromatography

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(HPLC)-grade methanol and acetonitrile were purchased from Fisher Scientific

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(Fairlawn, NJ). 3-nitrobenzonitrile (3-NBN) was purchased from Acros Organics

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(Geel, Belgium), 1,2-dicyanobenzene (1,2-DCB) was purchased from Sigma-Aldrich

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(St. Louis, MO). 3-NBN and 1,2-DCB are two common matrices used in LSI and inlet

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ionization. Standard samples used in experiments were all diluted in methanol−water

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(1:1 v/v). 3-NBN and 1,2-DCB were dissolved in acetonitrile, NFR was dissolved in

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water. All matrixes were prepared to a concentration of 0.1 mg/µL.

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Biological samples and materials. Met-Arg-Phe-Ala (MRFA, MW 523.65),

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Gly-Pro-Arg-Pro (GPRP, MW 425.48), angiotensin I (MW 1374.54), angiotensin II

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(MW 1046.18) and bradykinin (MW 1060.22), cytochrome c and myoglobin were all

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purchased from Sigma-Aldrich (St. Louis, MO). All peptides and proteins were

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diluted in methanol-water (1:1 v/v) with addition of 1% acetic acid. Urine was 7



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donated by a healthy volunteer in our lab. Blood and brain slices were obtained from

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experimental mice (C57BL/6 mice, 2 to 3 months old) in our university. The mice

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experimental studies were approved by Animal Care and Use Committee of Beijing

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Institute of Technology. Mice were decapitated and whole blood was collected in

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heparinized tubes, while its whole brain was harvested and flushed with saline for 5

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minutes to remove blood cells. The clean brain was stored in solution of 4%

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paraformaldehyde (PFA) for two days and then transferred to 30% sucrose solution at

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4 ℃. The dehydrated brains were sliced into 30 µm thick sections from coronal brain

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section in sequence using a Leica CM1850 cryostat (Leica Microsystems Inc.,

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Bannockburn, IL).

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2.3 Sample preparation

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Prior to the AP-LSI mass spectrometry analysis, a simple sample preparation

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step was needed. As shown in Figure 1b, a stainless-steel plate was used as the sample

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holder in this work. Glass slides were also tested, but no obvious differences were

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observed. Matrix solution in a volume of 10 µL was firstly loaded on the substrate

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plate using a pipette, followed by dropping 10 µL analyte solution into the matrix

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solution. After dried at room temperature, the analyte-matrix mixture was subjected to

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the laser ablation, and then analyzed by the miniature mass spectrometer.

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To prepare tissue sections, 10 µL purified water was firstly dropped onto the

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sample plate, and then a piece of brain section was laid onto it. After relaxing and

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tiling the tissue section in water condition, absorbent paper was then used to dry out

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the section. Finally, 10 µL matrix solution was spread over the tissue section and

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waited to dry at room temperature.

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3. Results and discussion

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3.1 Optimization and characterization of the AP-LSI

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As an atmospheric pressure ionization source, AP-LSI here involves analyte

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desorption, molecule transfer to the MS inlet, and ionization in the capillary inlet.

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Desorption and transfer efficiency of analytes depends on the choice of matrix, laser

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pulse energy and frequency, as well as the distance between the laser focus spot and

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the MS inlet (d and h shown in Figure 1b). The ionization efficiency of LSI is relevant

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to many factors, including the choice of matrix, temperature of the inlet capillary,

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vacuum pressure. In this study, LSI was performed on an miniaturized mass

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spectrometer, which has fixed inlet capillary and vacuum pressure. To transfer

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samples as much as possible into the capillary inlet and achieve a higher ionization

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efficiency, the choice of matrix, the relative position of sample spot to MS inlet, laser

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energy and frequency were optimized, and details could be found in the supporting

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information. After optimization, 3-NBN was selected as the LSI matrix, laser pulse

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energy and frequency were chosen to be 1.2 mJ/pulse and 10 Hz. Since 3-NBN does

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not have strong absorption in the IR region, it is believed that thermal heating might

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be the key effect of the IR laser on sample desorption. The vertical (h) and horizon (d)

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distance between laser spot and the mini-MS inlet were optimized as 5 mm and 6 mm,

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respectively. These optimized parameters were used in later studies in this work,

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otherwise specified.

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After optimization, sensitivity of the system was characterized using reserpine

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and MRFA. In the experiments, Samples of different concentrations were loaded on

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the sample holder, ion intensities were recorded in terms of analyte absolute amounts.

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Analyte absolute amount was defined and calculated as the absolute amount of

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analyte distributed in the ablated laser spot (details could be found in the Supporting

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Information). Figure 2a and 2c plot the linear range of quantitation curves of reserpine

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and MRFA, respectively. Seven parallel experiments were carried out in each

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measurement to minimize the sample non-uniform distribution effects. As shown in

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Figure 2b and 2d, limit of detections (LOD) of 50 pg and 200 pg in absolute amount 9



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were obtained for reserpine and MRFA, respectively. A signal-to-noise ratio (SNR) of

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SNR>5 was maintained in the LOD experiments.

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The capability of this AP-LSI mini MS in analyzing larger biomolecules were

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then explored. First, peptides including GPRP, bradykinin, angiotensin I and

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angiotensin II were tested. The amount of sample loaded in a laser spot area was ~1

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ng for each of these peptides. Mass spectra of these peptides were obtained and

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plotted in Figure 3. The mass spectrum of GPRP (Figure 3a) was dominated by singly

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charged protonated ion ([M+H]+); while multiply charged ions ([M+2H]2+ and/or

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[M+3H]3+) could also be observed or even dominated in the mass spectra of

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bradykinin, angiotensin I and angiotensin II. Similar to MAI, multiply charged ions

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were produced by AP-LSI in this system, which is believed to be attributed to the

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matrix and conditions the matrix particles experience in traversing the inlet tube.45,50

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Similar phenomenon was also observed by Sarah Trimpin group in their matrix

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assisted ionization (MAI) methods using 3-NBN as matrix.40,55-57 Experiments have

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also been carried out by applying different extraction voltages on the sample holder,

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and no obvious differences in terms of intensity and multiply charged ions were

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observed. Results confirm that ions were not formed during the laser ablation (refer to

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Supporting Information for details). In addition, a few fragment ions (b- and y- ions)

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were also observed in the mass spectra of bradykinin, angiotensin I, and angiotensin II

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with relative low abundance. Laser pulse energy and frequency were also tuned in this

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experiment, and it was found that the appearance and relative intensity ratios of these

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fragment ions were independent of laser parameters. Similar results were obtained

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when using different matrices. It is concluded that these fragment ions were not

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resulted from laser ablation, but very likely a consequence of in source fragmentation

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happened during ion transfer in the MS inlet.

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The multiple charged ions observed in our AP-IR-LSI mass spectra are similar to

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those acquired using electrospray ionization (ESI) sources, which is especially

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beneficial for our miniature mass spectrometer. Typically, the mass range of a

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miniature mass spectrometer is not as wide as lab-scale MS instruments, and larger 10


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biomolecules could be analyzed by lowering their mass-to-charge ratios (m/z) with

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multiple charges. Therefore, larger molecules, for example proteins, were expected to

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be able to be analyzed within the mass range of our mini-MS. Cytochrome c and

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myoglobin were used to demonstrate instrument capability of protein analyses.

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Following the same sample preparation procedures, 10 µL sample solutions (100

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µg/mL) were loaded onto the sample plate and mixed with the matrix. After the

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sample mixture dried, pulsed laser was then applied to conduct MS analysis. Figure 4

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shows the mass spectra of these two proteins. Multiply charged ions were well

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observed in the mass spectra, which show similar feature as that of conventional ESI

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mass spectra.

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3.2 Direct analyses of drugs in blood and urine

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Biological samples, such as blood and urine, have very complex matrix. Direct

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analysis of urine and blood is a great challenge for ESI-MS. Therefore, complicated

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sample pretreatment procedures are conventionally performed before MS or LC-MS

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analysis. However, in a typical application scenario of a miniature mass spectrometer,

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the sample pretreatment procedure or equipment are not desired and available.

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Therefore, a mini mass spectrometer normally needs to handle these complex samples

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directly. The capability of analyzing target analyte in complex biological samples

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with minimum sample pretreatment is then demonstrated using this AP-IR-LSI mini

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mass spectrometer. In the experiments, ciprofloxacin and reserpine were spiked into

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urine and whole blood, respectively. Prior to analysis, the urine stock solution was

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diluted two times and the blood stock solution was diluted four times in volume using

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methanol-water (1:1 v/v) as solvent. Then these samples were loaded onto sample

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plates and suspected to laser ablation after dried. Ciprofloxacin in urine and reserpine

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in blood could be directly detected with the mass spectra shown in Figure 5. Figure 5a

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and 5b are mass spectra obtained from ciprofloxacin in urine with an absolute amount

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of 100 ng and reserpine in blood with an absolute amount of 400 ng, respectively.

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Tandem MS was also performed using collision induced dissociation (CID) as shown

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in Figure 5c and 5d to confirm the results. 11



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3.3 Tissue analysis

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As a solid sample, tissue is another important type of biological sample. Lab scale

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MALDI-MS has been widely applied in tissue analysis, especially for tissue imaging

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in biomedical applications.58-60 By coupling with high-resolution mass spectrometers,

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LSI has also been used for imaging of mouse brain and other tissues samples.48,61,62 In

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this study, mouse brain tissue slices were analyzed using the AP-LSI mini MS system.

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Under the optimized working condition with a laser energy of 1.4 mJ/pulse, lipids in

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mouse brain were observed as shown in Figure 6. In the experiments, mass spectra

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with different relative abundances of m/z 735 and 789 were observed when analyzing

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different points on the tissue section. By exploring the relative abundance difference

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of mass peaks with their corresponding positions of tissue section, we found that

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higher relative abundance of m/z 735 (PC 32:0) resulted from grey matter and higher

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relative abundance of m/z 789 (PC 36:1) abundance was responsible for white matter.

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The results are consistent with that reported by other groups previously,63,64 who also

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revealed that PC 32:0 abundance is responsible for grey matter and PC 36:1

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abundance is associated with white matter. As noticed, the spatial resolution of the

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current AP-LSI is only about 0.6 mm. Issues, such as spatial resolution, MS resolution

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and carryover effects need to be solved, before this system is ready to perform MS

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imaging experiments.

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4. Conclusions

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In this study, an AP-IR-LSI miniature mass spectrometer was developed. Using

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3-NBN as matrix and under optimized working conditions, an LOD of 200 pg in

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absolute amount could be achieved for standard samples. The rapid analyses of

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biological samples, including peptides, proteins, drugs in urine and blood, tissue

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sections were demonstrated using the system with minimum sample preparations. By

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coupling with an IR laser, the instrument would actually lose portability. However,

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this laserspray ionization method could greatly simplify the ionization and sample

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preparation procedure, especially for complex samples. The AP-IR-LSI mini-MS has

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capability of direct analyzing complex samples on spot and is potentially feasible for

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environmental monitoring, point-of-care diagnostic testing and rapid MS imaging.

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Acknowledgements This work was supported by NNSF (21475010, 61635003), BNSF (16L00065) and State Key Laboratory of Explosion Science and Technology (YBKT16-17).

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Page 17 of 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


1

Figure 1

2 3

Figure 1. AP-LSI miniature mass spectrometer setup. Three-dimensional setup of the

4

instrument (top); Schematic plot of sample preparation and laser ablation.

5

17



1

Figure 2

Reserpine (LOD 50 pg)

75 50 25 0

y = 6.0943x + 1253.3 R² = 0.9943 0

1000

1500

2000

2500

absolute amount (pg)

(c)

2

500

MRFA (LOD 200 pg)

50

y = 0.6726 x + 3.6949 R² = 0.9973

25 0

0

1000

2000

3000

4000

609

100

Reserpine (50 pg) 75 50

S/N > 5

25 0 200

300

400

5000

500

600

700

800

m/z

(d)

100 75

relative abundance (%)

(b)

100



(a)


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 18 of 23

100

MRFA (200 pg) 524 75 50

S/N > 5

25 0 200

300

absolute amount (pg)

400

500

600

700

800

m/z

3

Figure 2. Standard sample analysis using the AP-LSI miniature mass spectrometer. (a)

4

Linear range of quantitation for reserpine; (b) mass spectrum of reserpine, 50 pg of

5

reserpine was presented in the laser spot; (c) Linear range of quantitation for MRFA;

6

(b) mass spectrum of MRFA, 200 pg of MRFA was presented in the laser spot;

7

18


Page 19 of 23

1

Figure 3

[M+H]+ 426

100

GPRP

(b)



(a)

75 50 25 0 200

300

400

500

100

[M+2H]2+ 531

Bradykinin

75 50

710

0 400

600

2

100

Angiotensin I

25

[M+3H]3+

75 50

0 400

[M+H]+ 1296

b 6+ 784

433

600

800

800

1000

1200

m/z (d)

[M+2H]2+ 648

1000

1200

1400


(c)

y8+ [M+H]+ 904 1061

y6+

25

m/z


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


100

[M+2H]2+ 524

Angiotensin II [M+H]+ 1047

75 50

b6+ 784

25 0 400

600

800

1000

m/z

m/z

3

Figure 3. Peptide analyses using the AP-LSI miniature mass spectrometer. Mass

4

spectra of (a) GPRP, (b) bradykinin, (c) angiotensin I and (d) angiotensin II.

5

19



1

Figure 4

100


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

12+ 13+ 1031 951

Cytochrome c

75

11+ 1125

15+ 825 14+ 884

50

(a)

10+ 1237

9+ 1374

16+ 17+ 773 728

25 0 100

14+ 1212

Myoglobin

15+ 16+ 1131 1061

75 17+ 19+ 18+ 998 883 21+ 943 80820+ 849

50 25 0 600

2

Page 20 of 23

700

800

13+ 1305

(b) 12+ 1414

900 1000 1100 1200 1300 1400 1500

m/z

3

Figure 4. Protein analyses using the AP-LSI miniature mass spectrometer. Mass

4

spectra of (a) cytochrome c and (b) myoglobin.

5

20


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1

Figure 5

2 3

Figure 5. Detection of drugs in complex matrices using the AP-LSI miniature mass

4

spectrometer. (a) Mass spectrum of ciprofloxacin in urine, 100 ng of ciprofloxacin

5

was presented in the laser spot; (b) the corresponding tandem mass spectrum of 100

6

ng ciprofloxacin in urine; (c) mass spectrum of reserpine in blood, 400 ng of reserpine

7

was presented in the laser spot; (b) the corresponding tandem mass spectrum of 400

8

ng reserpine in blood.

9

21



1

Figure 6

100


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 22 of 23

735 [PC(32:0)+H]+

75 50

761 [PC(34:1)+H]+ 773 [PG(36:3)+H]+ 789 [PC(36:1)+H]+ 799 [PG(38:4)+H]+

grey matter 833 [PI(34:3)+H]+

25 0 100 75 50

[PC(34:1)+H]+ 761 789 [PC(36:1)+H]+ 799 [PG(38:4)+H]+ [PC(32:0)+H]+ 735 833 [PI(34:3)+H]+

white matter

25 0 300 400 500 600 700 800 900 1000

2

m/z

3

Figure 6. Tissue section analyses using the AP-LSI miniature mass spectrometer. (a)

4

Mass spectrum of grey matter in a mouse brain tissue section; (b) mass spectrum of

5

white matter in a mouse brain tissue section.

6

7

8

22


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1

2

Only for TOC

3 4

23


Direct Biological Sample Analyses by Laserspray Ionization Miniature

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