Development and Characterization of A Linear Matrix-assisted Laser ...

CHINESE JOURNAL OF ANALYTICAL CHEMISTRY Volume 46, Issue 4, April 2018 Online English edition of the Chinese language journal

Cite this article as: Chinese J. Anal. Chem., 2018, 46(4): 463–470

RESEARCH PAPER

Development and Characterization of A Linear Matrix-assisted Laser Desorption Ionization Mass Spectrometer YU Jia-Jun1, LIU Ping3, ZENG Zhen1, CHEN Ying1, GAO Wei1,2, LI Mei1,2, WANG Chen-Guang4, HUANG Zheng-Xu1,2, ZHOU Zhen1,2, LI Lei1,2,*

1

Institute of Mass Spectrometer and Atmospheric Environment, Jinan University, Guangzhou 510632, China Guangdong Provincial Engineering Research Center for On-line Source Apportionment System of Air Pollution, Guangzhou 510632, China 3 Guangzhou HeXin Instrument Co., LTD, Guangzhou 510530, China 4 Guangzhou Institute of Energy Conversion, Chinese Academy of Sciences, Guangzhou 510640, China 2

Abstract:

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is an important

analytical technique for biological macromolecules, such as proteins, peptides and nucleic acid, especially in the field of microbial identification. On the basis of previous study, a linear MALDI-TOF MS was been designed and assembled for biological applications. The instrument comprised a vacuum system, a vacuum fast sample introduction system, an optical system, a time-of-flight mass analyzer, an ion source, a data acquisition system and an electronic control system. The ion source adopted two-stage source acceleration, delayed extraction and dynamic pulse focusing technique. The time-of-flight distance of field-free drift region was about 1 m. The optical system adopted a solid-state laser with adjustable frequency of 1–2000 Hz and spots of 20–100 μm. The angle of incidence laser was controlled at 5°. A series of experiments were carried out to further evaluate the instrument performances. It could not only analyze the samples more than 199 kDa, but also achieve isotope resolution at 1000–3000 Da and up to 900 (FWHM) at 5000–17000 Da. The minimum detectable concentration of gramicidin was 10 amol μL‒1, absolute sensitivity reached up to 2.56 amol. Independent detection of saliva samples from different targets showed that the instrument had higher producibility. We identified Escherichia coli and Shigella spp., which are two common bacteria but difficult to be differentiated by mass spectrometry, showing its potential identification for clinical microorganism. In summary, this instrument can play a role on clinical examination in the near future. Key Words:

Matrix-assisted laser desorption ionization; Linear time-of-flight mass spectrometer; Development; Performance

characterization

1

Introduction

In the late 1980s, the emergence of two ionization techniques, electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI), provided important and powerful techniques for the analysis of macromolecules[1,2]. Since then, significant progress has been made for ESI

technology. An ESI ion source can be easily used in combination with various mass analyzers. Until now, ESI has remained a valid tool for the qualitative and quantitative determination of macromolecules[3,4]. Recently, MALDI has markedly improved with developments in laser technology, high-speed data acquisition, ion detection, and matrix technology. The modern MALDI instrument has high

________________________ Received 11 November 2017; Accepted 1 December 2017 *Corresponding author. E-mail: [email protected] This work was supported by the Guangdong Applied Science and Technology Research and Development, China (No. 2015B020236003), and the Fundamental Research Funds for the Central Universities of China (No. 21616341). Copyright © 2018, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved. DOI: 10.1016/S1872-2040(17)61077-6

YU Jia-Jun et al. / Chinese Journal of Analytical Chemistry, 2018, 46(4): 463–470

resolution, high sensitivity, a wide mass range, and the capability for quantitation[5]. The combination of MALDI ionization source with time-offlight mass spectrometry (TOF MS), is a very successful application of MS. There are three types of MALDI-TOF MS: linear, reflection, and TOF/TOF[6–8]. Linear instruments own the simplest structure with some very important technical advantages. In particular, metastable protein macromolecules are prone to fragmentation in an electric field or in flight during MALDI ionization. Although this fragmentation can result in incomplete protein analysis[9], it does not affect the analytical results of macromolecular ions in linear MALDI-TOF MS. Thus, the mass range of analysis using linear MALDI-TOF MS is extended to millions or even tens of millions of Daltons (Da)[10]. This is much higher than those instruments using reflection or TOF/TOF. Therefore, it has many advantages in the detection of macromolecules. In recent years, MALDI-TOF MS has gradually become an important technique to analyze biological macromolecules such as proteins, peptides, microorganisms and nucleic acids[11–13]. To identify microorganisms, the mass spectra of microorganisms from clinical samples were compared to a standard protein fingerprint database[14]. Compared with traditional microbial identification techniques such as phenotyping, biochemical, and luminescence methods, this method has advantages in terms of identification speed, accuracy of results, technical costs, and requirements for operational staff[15,16]. The technical features of linear MALDI-TOF MS include high sensitivity, a wide mass range, and moderate resolution/mass accuracy. In fact, in the field of microbial identification and nucleic acid detection, linear MALDI-TOF MS has reached the clinical application stage and is clinically approved in the United States, Europe, and China. This makes it the most widely used MALDI-TOF MS[17,18]. Expanding MALDI-TOF MS to quantitative analysis has been challenging. Vestal et al[19] performed a series of technical improvements on linear MALDI-TOF MS instrument, and the ability of the instrument in quantitative analysis was improved considerably. The quantitative determination of human glycated hemoglobin using this instrument illustrated that linear MALDI-TOF MS had a wide range of applications for quantitative clinical measurements[20]. The linear MALDI-TOF MS will be applied in clinical practice widely with future improvement in instrumentation and application. In this work, we independently developed a linear MALDI-TOF MS system based on previrous work and special application requirements. In this system, a unique ion source structure as well as delayed extraction and dynamic pulse focusing were used to achieve high resolution over a wide mass range. We used a vertical laser beam path to minimize the initial analyte velocity and position dispersion. This design

ensured good resolution and detection performance. The use of a long-life and high frequency solid-state laser increased the detection speed of the instrument, which was more suitable for high throughput clinical detection. Here, we described the main structure, the key technologies, and the basic performance of the system, and further evaluated the system. This instrument would play a role in future clinical applications.

2 2.1

Experimental Principle and structure of instrument

As shown in Fig.1, the homemade MALDI-TOF MS included a vacuum system, a vacuum fast sample introduction system, an optical system, a TOF mass analyzer, a data acquisition system, and an electronic control system. The vacuum of the system was maintained by two turbomolecular pumps and a diaphragm pump with a vacuum pressure of about 4 × 10−5 Pa. MALDI-TOF MS requires a high vacuum to maintain when changing samples. Thus, a threedimensional motion platform was designed. X-Y direction of the platform was used for the plane movements of a sample target, and Z direction of the platform was used for rapid sample loading and switching. An enclosed cavity was formed between the sample target and the cover of the sample-changing chamber after the sample plate was moved to the top using the Z-direction mechanical device. Subsequently, the pumping and air-release lines were used to realize free switching between air/vacuum environments in the independent cavity. This offered rapid switching of the sample target. It only took 2 min to change a plate. The TOF mass analyzer consisted of an ion source, a field-free flight tube, and a detector. The field-free flight distance of the instrument was 1 m and included a homemade high-performance module ion detector. To obtain fast on-board signal processing, a high-speed data acquisition card with 2 GSa s-1 sampling rate and a 500 MHz bandwidth was used for data acquisition. 2.2

Ion source

Figure 2 shows a schematic diagram of the structure of the ion source. The main structure of the ion source was two-field sources consisting of two accelerating field plates, an ion focusing lens consisting of a group of lenses, a group of deflection electrodes, and an optical path-introducing device. The lenses and deflection plates were used to focus ion beam and deflecting of the matrix ions, respectively. Here, a DC voltage of more than 20 kV was applied to the sample target plate and the first accelerating plate. After the laser irradiated the sample, there was a 100-ns delay before the positive and negative pulses were applied on the sample target plate and the first accelerating plate by the circuit, respectively. The

YU Jia-Jun et al. / Chinese Journal of Analytical Chemistry, 2018, 46(4): 463–470

Fig.1

Fig.2

Appearance (A) and schematic diagram (B) of homemade matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF MS) system

Schematic illustration of homemade ion source

ions after focusing then flied to the detector through a field free region. The delayed extraction technique could dramatically enhance the mass resolution of MALDI-TOF MS[21], and the dynamic pulse focusing method could achieve a relatively high mass resolution over a wide mass range[22]. The ion velocity dispersion greatly affects the sensitivity and resolution of the instrument. This is because the ion cloud produced by the laser bombardment of the sample has an initial velocity, whose magnitude and direction are directly related to the incident angle of the laser and the energy intensity[23,24]. Therefore, the incident direction of the laser light needs to be perpendicular to the sample target to minimize the dispersion of the initial position and velocity of the ion cloud. The angle between the laser incident light and the vertical line to the sample target was controlled within 5° via the optical path-introducing device. This special design of the ion source and optics minimized initial ion dispersion and improved resolution of the instrument. 2.3

Optical structure

Most linear MALDI-TOF MS systems use nitrogen lasers as ionization sources. Nitrogen laser has a uniform spot and

low cost; however, due to its inherent limitations, the repetition frequency is typically only a few tens of hertz with a short lifetime. Solid-state lasers have gradually replaced nitrogen lasers because they are small, stable, and have excellent beam quality. The high-frequency solid-state laser used here was 343 nm with an adjustable operating frequency of 1–2000 Hz. Its high repetition frequency could increase the speed of sample analysis and dramatically improve the capability of the instrument for MS imaging, leading to the enhancement of the quantitative capability of the instrument. The laser beam was focused on the sample target surface through a series of optical components. The size of a laser spot and laser beam energy could be adjusted from 20–100 μm and 0–80 μJ, respectively, which improved the efficiency of the analysis of different substances. Furthermore, the laser beam was also synchronized to trigger high-voltage pulses and data acquisition cards as a starting point for data acquisition. In addition, an optical imaging system was designed to view and confirm the status of the target in real time, by which the crystallization status and ionization status of the sample could be observed. 2.4

Samples and reagents

The samples used to characterize the performance of the instrument included a peptide calibration standard (# 206195, Bruker Daltonics, Bremen, Germany), protein mix standards (#206355, Bruker Daltonics, Bremen, Germany), bovine serum albumin (BSA, 66431 Da, 100 pmol μL–1, SigmaAldrich, Shanghai, China), gramicidin S (1141.5 Da, 104 pmol μL–1, Institute of Energy Problems for Chemical Physics (Branch) Russian Academy of Sciences), saliva samples (self-collected and diluted 10-fold with pure water), and Escherichia coli and Shigella flexner standard strains (Guangdong Institute of Microbiology, China). The standard solutions of protein and peptide standards at different concentrations were prepared. Saturated α-cyano-4-

YU Jia-Jun et al. / Chinese Journal of Analytical Chemistry, 2018, 46(4): 463–470

hydroxycinnamic acid (CHCA, Sigma) and sinapinic acid (SA, Sigma) were selected as the matrixes, and the sample and matrix were mixed in a 1:1 volume ratio followed by a thin layer method for sample deposition. Each target spot was covered with 1.0 μL of sample, which was dried and covered by 1.0 μL of matrix. Subsequently, the target plate was air dried for future analysis. For bacterial samples, a small amount of liquid bacteria culture was picked with a sterile toothpick and smeared evenly on the target spot. After slight drying, the cells were covered with 1.0 μL of CHCA matrix and completely dried for MS analysis.

3

Results and discussion

3.1

Resolution

Figure 3 shows the MALDI-TOF mass spectra of peptide calibration standards. The peaks of angiotensin II (1046.5 Da), angiotensin II (1296.7 Da), substance P (1347.7 Da), bombesin (1619.8 Da), adrenocorticotropic hormone 1–17 (ACTH clip 1–17, 2093.1 Da), and adrenocorticotropic hormone 18–39 (ACTH clip 18–39, 2465.2 Da) could be isotopically resolved. The full width at half maximum (FWHM) calculation showed that the resolutions of single isotope peaks were 5070, 4590, 5545, 6920, 5140 and 6950, respectively. The mass spectra of protein mixed standard are shown in Fig.4. The resolution of insulin (5734 Da), cytochrome C

Fig.3

MALDI-TOF mass spectra of peptide standard solution

Fig.4

MALDI-TOF mass spectra of protein standard solution

YU Jia-Jun et al. / Chinese Journal of Analytical Chemistry, 2018, 46(4): 463–470

(12361 Da) and myoglobin (16952 Da) could be up to 900 (FWHM). The resolution of this MALDI-TOF MS system reached the technical requirement of the same type of commercial instruments, and a relatively higher resolution was achieved over a mass range of 1000 to 20000 Da. This was suitable for the detection of most biological macromolecules such as peptides, some proteins and microorganisms. 3.2

Mass range

An important advantage of linear MALDI-TOF MS is the ability to efficiently analyze high-mass analytes. Here, BSA (66456.5 Da, 30 pmol μL–1) deposited on the SA matrix was used to test the mass range of detection. Figure 5 shows the sample spectrum. In addition to the parent ion peak, a BSA trimer with a mass of 199291 Da was detected, confirming that our instrument could detect analytes with a mass up to 199 kDa. Studies with larger analytes (MS > 199 kDa) are underway. The current detection and identification of microorganisms by MALDI-TOF MS mainly focuses on the analysis of their ribosomal proteins, whose masses are mainly distributed from 2.0 kDa to 20 kDa[14,25,26]. Therefore, the developed MALDI-TOF MS instrument in this work could meet the requirements of microbial identification in terms of mass range. 3.3

Sensitivity

To determine limit of detection of instrument, a series of

Fig.5

Fig.6

standard solutions of gramicidin S were prepared: 10000, 1000, 500, 100, 50 and 10 amol μL–1, CHCA was used as matrix, and the thin layer method was used for sample deposition. The samples were air dried before the measurement. As shown in Fig.6, the instrument could effectively detect gramicidin S standard samples at different concentrations. Strong and stable peaks were observed, and the detection limit was as low as 10 amol μL–1 (S/N > 3). Only a small fraction of the sample was actually consumed during MALDI-TOF analysis. Thus, the absolute sensitivity (S) of the instrument could be calculated based on the equation (1): S = CVA2/2A1 (1) where, C is the concentration of the sample standard solution, V is the droplet volume of the mixture of matrix and sample spotted at each target, A1 is the distribution area of the samplematrix mixture co-crystallized at a single target, and A2 is the equivalent total area of the sample that is actually desorbed when the laser bombards the target. The area of the sample desorbed by laser desorption is approximately equal to the size of the focused spot on the target. Here, C was 10 amol μL–1 of standard gramicidin S, and the mixed sample volume for spotting (V) was 1.0 μL. The laser spot diameter was about 80 μm, and each spectrum was accumulated 320 times. If the sample at each target bombarded by the laser was completely consumed, then the equivalent desorbed sample area was about 1.61 mm2. However, the diameter of the sample at a single target as measured by a microscope was about 2 mm, i.e., a co-crystallization area of 3.14 mm2 (Fig.7). Thus, based on equation (1), the minimum sample consumption for the detection of gramicidin S was about 2.56 amol.

MALDI-TOF mass spectrum of bovine serum albumin (BSA, 100 pmol μL–1)

MALDI-TOF mass spectra of gramicidin S with different concentrations

Fig.7

Crystalline state of the mixture of gramicidin S (10 amol μL–1) and CHCA matrix on the sample plate

YU Jia-Jun et al. / Chinese Journal of Analytical Chemistry, 2018, 46(4): 463–470

3.4

Reproducibility and mass accuracy

The repeatability of MS instrument is important to identify analytes. Saliva sample contains mucin, mucopolysaccharide, salivary amylase, lysozyme, immunoglobulin (lgA, lgG and lgM), blood group substances (a, B and H), urea, uric acid, free amino acids, inorganic ions and other ingredients, and thus is an ideal candidate to verify instrument reproducibility. Here, fresh saliva samples were collected from healthy human volunteers and diluted with pure water at a ratio of 1:10 (V/V), and mixed evenly by shaking. The same volume of saliva and CHCA matrix were collected, and a thin layer cover method was used for sample deposition. Individual spectrum was acquired on four different adjacent targets. The spectrum at each target was accumulated 320 times. As shown in Fig.8, the spectra of four saliva samples were mainly distributed from 2000–6000 Da, and stable and strong signal peaks were achieved for all mass ranges. The inset is an enlarged spectrum from 1200–4000 Da. This primarily showed the large number of peaks with low intensities. The characteristics of the saliva spectra obtained from four different targets were essentially the same, indicating that this homemade instrument had excellent reproducibility in the analysis of different targets. To further evaluate the mass accuracy of the instrument, internal and external standard methods were adopted for mass calibration using a mixture of peptides and proteins. The results showed that the mass accuracy could be controlled within 80 ppm (substance P, 1347.7 Da) and 150 ppm (cytochrome C, 12361 Da), respectively, with the internal standard method. The external standard method allowed a mass accuracy of 150 ppm (substance P, 1347.7 Da) and 200 ppm (cytochrome C, 12361 Da), respectively. These results indicated that our instrument can meet the measurement requirements of variety of samples.

Fig.8

3.5

Bacterial analysis

To verify the practical utility of this instrument in the field of microbial identification, Escherichia coli and Shigella flexneri were selected for measurement and identification. Escherichia coli and Shigella flexneri have been found to exhibit a similar genetic background including their ribosomal proteins[14,27]. Thus, it is difficult to distinguish them using MALDI-TOF MS [28]. Figure 9 shows the mass spectra of Escherichia coli and Shigella flexneri acquired from the homemade MALDI-TOF MS system. Although the major protein peaks of two species were basically the same, the peak intensities had a significant difference. The two species were identified using commercially available software (MicroID (http://www.microid.net/MicroID.aspx); Fig.9 inset). Both bacteria were successfully identified with a score of 9 points or higher (a score of 8 points or higher indicates that the result is reliable, a score of 6–8 points indicates that the result can be used as a reference, and a score below 6 points indicates that there is no identification result or the result is not reliable). The instrument could distinguish between two bacterial species. The results suggest that our instrument can be used to identify clinically relevant microbes.

4

Conclusions

A linear MALDI-TOF MS system was independently developed in this work. A long-life high-frequency solid-state laser was used to offer an adjustable frequency, pulse energy, and spot size. The unique ion source structure and optical path system minimized the initial velocity and position dispersion of analytic ions, which ensured high mass resolution over a wide mass range. The validation with peptides and protein standards showed that the instrument could efficiently detect

Four independent MALDI-TOF mass spectra of human saliva samples

YU Jia-Jun et al. / Chinese Journal of Analytical Chemistry, 2018, 46(4): 463–470

Fig.9

MALDI-TOF mass spectra of Escherichia coli (A) and Shigella flexneri (B)

target analytes up to over 199 kDa with good resolution over the full mass range up to 20000 Da. The experiment results with gramicidin S and saliva samples demonstrated the instrument possessed low detection limits and high reproducibility. Escherichia coli and Shigella flexneri were easily distinguished with this system, suggesting its potential for identifying clinically relevant microbes. Our future work will focus on improving stability of the instrument and data analysis algorithms to make it be effectively applied in clinical diagnostics.

References

[10] Spengler B, Kirsch D, Kaufmann R. J. Phys. Chem., 1992, 96(24): 9678–9684 [11] Tran B Q, Barton C, Feng J H, Sandjong A, Yoon S H, Awasthi S, Liang T, Khan M M, Kilgour D P A, Goodlett D R, Goo Y A. J. Proteomics, 2016, 134: 93–101 [12] Rodrigo M A M, Zitka O, Krizkova S, Moulick A, Adam V, Kizek R. J. Pharm. Biomed. Anal., 2014, 95: 245–255 [13] Meyer S, Vollmert C, Trost N, Sigurdardottir S, Portmann C, Gottschalk J, Ries J, Markovic A, Infanti L, Buser A. Br. J. Haematol., 2016, 174(4): 624–636 [14] Fenselau C, Demirev P A. Mass Spectrom. Rev., 2001, 20(4): 157‒171 [15] Saffert R T, Cunningham S A, Ihde S M, Jobe K E M,

[1]

Fenn J B, Mann M, Meng C K, Wong S F, Whitehouse C M. Science, 1989, 246(4926): 64–71

Mandrekar J, Patel R. J. Clin. Microbiol., 2011, 49(3): 887–892 [16] Davies A P, Reid M, Hadfield S J, Johnston S, Mikhail J, Harris

[2]

Karas M, Hillenkamp F. Anal. Chem., 1988, 60(20): 2299–2301

L G, Jenkinson H F, Berry N, Lewis A M, El-Bouri K, Mack D.

[3]

Zhu X Q, Huang Z X, Gao W, Li X, Li L, Zhu H, Mo T, Huang

J. Clin. Microbiol., 2012, 50(12): 4087–4090

[4]

Wang Z H, Chen Y H, Xu J, Chen H L, Zhou X, Xiao X H,

B, Zhou Z. J. Agric. Food Chem., 2016, 64(27): 5614‒5619 Ping F, He J M, Zeper A, Zhang R P. Chinese J. Anal.Chem., 2017, 45(5): 654‒661

[17] Mather C A, Rivera S F, Butler-Wu S M. J. Clin. Microbiol., 2014, 52(1): 130–138 [18] Ferrand J, Hochard H, Girard V, Aissa N, Bogard B, Alauzet C, Lozniewski A. J. Clin. Microbiol., 2016, 54(3): 782–784

[5]

Vestal M L. J. Am. Soc. Mass Spectrom., 2011, 22(6): 953‒959

[19] Vestal M L. Clin. Chem., 2016, 62(1): 20–23

[6]

Prentice B M, Chumbley C W, Hachey B C, Norris J L,

[20] Hattan S J, Parker K C, Vestal M L, Yang J Y, Herold D A,

Caprioli R M. Anal. Chem., 2016, 88(19): 9780–9788 [7]

[8]

Medzihradszky K F, Campbell J M, Baldwin M A, Falick A M,

532–541

Juhasz P, Vestal M L, Burlingame A L. Anal. Chem., 2000,

[21] Brown R S, Lennon J J. Anal. Chem., 1995, 67(13): 1998–2003

72(3): 552–558

[22] Franzen J. Int. J. Mass Spectrom. Ion. Processes., 1997, 164(1):

Vestal M L, Hayden K. Int. J. Mass spectrom., 2007, 268(2): 83–92

[9]

Duncan M W. J. Am. Soc. Mass Spectrom., 2016, 27(3):

Karas M, Kruger R. Chem. Rev., 2003, 103(2): 427–440

19–34 [23] Bökelmann V, Spengler B, Kaufmann R. Eur. J. Mass Spectrom., 1995, 1(1): 81–93

YU Jia-Jun et al. / Chinese Journal of Analytical Chemistry, 2018, 46(4): 463–470

[24] Aksouh F, Chaurand P, Deprun C, Della-Negra S, Beyec Y L,

221–242

Hoyes J, Pinho R R. Rapid Commun. Mass Spectrom., 1995,

[26] Sauer S, Kliem M. Nat. Rev. Microbiol., 2010, 8(1): 74–82

9(6): 515–518

[27] Arnold R J, Reilly J P. Anal. Biochem., 1999, 269(1): 105–112

[25] Keys C J, Dare D J, Sutton H, Wells G, Lunt M, McKenna T, McDowall M, Shah H N. Infect. Genet. Evol., 2004, 4(3):

[28] Khot P D, Fisher M A. J. Clin. Microbiol., 2013, 51(11): 3711–3716