MSc Chemistry
Analytical Sciences
Master thesis
DEVELOPMENT OF BIOLOGICAL COMPOUND ARRAYS, EMBEDDED
IN TISSUE MIMETIC MATERIALS FOR TESTING THE SENSITIVITY AND
DETECTION LIMITS OF MASS SPECTROMETRY IMAGING
TECHNIQUES
by
Skandalaki Eleni
February 2014
Supervisor:
Dhr. Dr. W.T. Kok
Daily Supervisor:
Prof.Dr.Ing. R. Heeren
Research was carried out at the group of Bio Molecular Imaging Mass Spectrometry (BIMS) at
AMOLF-FOM institute
Eleni Skandalaki
UvA-AMOLF
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Abstract
Mass spectrometry (MS) and the techniques based on it, are significant tools for researchers
due to their high sensitivity, accuracy and capability of detecting and distinguishing
compounds of high masses. Ergo, techniques depending on MS are suitable for applications
in disciplines like chemistry, and biochemistry. One of the innovative techniques depending
on MS that is widely employed in biochemistry is mass spectrometry imaging (MSI). With
MSI, it is possible to localize, detect and visualize compounds of interest simultaneously; this
is why it was considered an appropriate technique to use in this project. In particular, by
using Maldi Assisted Laser Desorption Ionization (MALDI) mass spectrometry imaging it was
attempted to create a quantification method for MALDI instruments, by developing test
arrays of lipids (1, 2-Dipalmitoyl-sn-glycero-3-phosphocholine, Sphingomyelin), peptides
(Substance P, Angiotensin II) and proteins (Bovine serum albumin, Trypsinogen) embedded in
tissue mimetic materials such as gelatin and Carboxymethyl cellulose salt (CMC) solution.
Even though the tissue mimetics appeared to mix with the compounds of interest, this did
not occur in the test arrays developed and the compounds could not be distinguished by
observing the images created after MSI analysis; therefore the creation of a quantification
method was not achieved. Despite that fact, it was realized that in the event of using
commercial hydrogels as tissue mimetics and different biological compounds for the
development of the arrays, a qualification method could be created. Another part of this
project included spatial resolution experiments with the biological compounds that are
already mentioned. The results of these experiments showed that the MALDI- TOF MS
instruments in the Bio Molecular Imaging Mass Spectrometry (BIMS) laboratory at AMOLFFOM institute have high sensitivity and high limit of detection.
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Table of Contents
Abstract ................................................................................................................................................... 2
Introduction............................................................................................................................................. 5
Mass spectrometry and Mass Spectrometry Imaging......................................................................... 5
Matrix Assisted Laser Desorption Ionization (MALDI)......................................................................... 6
Principles of Time of Flight (TOF) MS .................................................................................................. 7
Principles of Secondary Mass Spectrometry (SIMS)............................................................................ 8
Compounds employed throughout the project .................................................................................. 8
Calibration lines ..................................................................................................................................... 12
Profiling of compounds of interest.................................................................................................... 13
DPPC calibration lines........................................................................................................................ 15
SM calibration lines ........................................................................................................................... 17
Peptide mixture calibration lines ...................................................................................................... 18
Protein mixture calibration lines ....................................................................................................... 19
An alternative normalization technique ........................................................................................... 20
Development of Test Arrays .................................................................................................................. 22
Choice of tissue mimetic materials ................................................................................................... 22
Choice and design of proper container ............................................................................................. 22
Sectioning .......................................................................................................................................... 23
Matrix application ............................................................................................................................. 24
MSI analysis in positive mode and Data Analysis .............................................................................. 25
MSI analysis in negative mode and Data analysis ............................................................................. 27
Principle Component Analysis (PCA) and Discriminant Analysis (DA)............................................... 28
Profiling of DPPC and SM arrays with Secondary Ionization Mass Spectrometry (SIMS) ................. 34
Spatial resolution experiments ............................................................................................................. 36
Sample preparation ........................................................................................................................... 36
Acquired Data from MSI analysis of DPPC ........................................................................................ 37
Acquired Data from MSI analysis of SM ............................................................................................ 38
Acquired Data from MSI analysis of Peptide Mixture -Substance p ................................................. 39
Acquired Data from MSI analysis for Angiotensin II.......................................................................... 40
Protein mixture- Sample preparation ............................................................................................... 42
Acquired Data from MSI analysis of BSA ........................................................................................... 42
Acquired Data from MSI analysis of Trypsinogen ............................................................................. 43
Results, discussion and suggestions ...................................................................................................... 45
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Suggestions........................................................................................................................................ 46
References ............................................................................................................................................. 47
Appendix................................................................................................................................................ 50
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Introduction
Mass spectrometry and Mass Spectrometry Imaging
“Mass spectrometry is the study of matter through the formation of gas-phase ions that are
detected and characterized by their mass and charge” according to Murray, Boyd, Eberlin,
Langley, Li & Naito (2013). Mass spectrometry and the techniques based on it, are significant
research tools that are widely utilized in chemistry, biology, physics, and have numerous
environmental, geological, biotechnological, pharmaceutical and clinical applications.
Furthermore, mass spectrometry is extensively used in these fields, because of its high
sensitivity, accuracy; resolution and wide mass range detection limit (Chughtai 2012).
One of the innovations that have been developed in the field of mass spectrometry due to
its technological and methodological benefits is mass spectrometry imaging (MSI). With MSI,
the visualization of the spatial distribution of different compounds based on their molecular
masses synchronously is possible (McDonnell & Heeren 2007, Caldwell & Caprioli 2005). For
this reason, MSI has several applications in pharmaceutical and biomedical studies and in
particular it has been used in many studies about tissue engineering with which this project
is related. In this project, Matrix Assisted Laser Desorption (MALDI) mass spectrometry
imaging was employed in the majority of experiments.
Aim of project
The objective of this project was to contribute in the on-going search of innovations in the
mass spectrometry imaging, by creating test arrays of different biological compounds such
as lipids, proteins and peptides embedded in tissue mimetic materials. The development of
such arrays could be used for the creation of a quantification method that could indicate the
sensitivity and the limit of detection of specific MSI techniques in different laboratories.
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Matrix Assisted Laser Desorption Ionization (MALDI)
Matrix Assisted Laser Desorption Ionization (MALDI) is the most widely utilized MSI
technique (Gode & Volmer 2013); it is a ‘’soft’’ ionization technique which has proved to be
an important analytical tool (Hillenkamp & Karas 2007). A technique is defined as ‘’soft’’
when the energy that is supplied to the samples is lower compared to electron ionization,
therefore the produced fragments are fewer and the obtained spectra are simple (Games
1978). MALDI was developed in the 1980s by F.Hillenkamp and M.Karas, and ever since it is a
powerful source for producing intact gas-phase ions from numerous large, labile and nonvolatile compounds. Furthermore, as it is suggested from the name of MALDI the matrix that
is applied on the sample is of tremendous significance because it determines the extent of
the ionization and desorption of the analyte of interest. In particular, the MALDI process
takes place in two parts. Firstly, the matrix solution is an aromatic acid dissolved in a mixture
of organic solvents and it should have the capability of absorbing at a specific laser
wavelength and remain stable under vacuum. The matrix is applied on the sample during the
sample preparation. While it is applied, the solvents employed for the dilution of the matrix
evaporate and it reacts with the sample leading to the formation of matrix crystals. The
second step of MALDI, takes place under vacuum in the mass spectrometer source. During
this stage, the sample and matrix co-crystals are “cut” by laser pulses that occur within a
short window of time. Afterwards the co-crystals are heated quickly by the irradiation that
the laser induces, to such an extent that localized sublimation occurs and intact analyte
enters the matrix plume. Throughout this process, ionization reactions take place, and the
mechanism that is presumed to be responsible for the ionization, is described according to
Hoffmann & Stroobant (2007) with “proton transfer in the solid phase before desorption or
gas-phase photon transfer in the expanding plume from photoionized matrix molecules”.
Lastly, an electrostatic field induces the acceleration of the ions that are in the gas phase in
the direction of the analyser. In the figure 1.1 there is a scheme depicting the principle of
MALDI (Hoffmann& Stroobant 2007).
Figure1.1 Principle of MALDI ionization, (Hoffmann& Stroobant 2007)
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In this project, the instruments used for the MSI analysis were the SYNAPTTM HDMS, Waters
Corporation, Milford, MA and Ultraflex III, Brucker Daltonics. These instruments are MALDI
tandem mass spectrometers, which are used for automated MS and MS/MS high throughput
detection and identification of peptides and proteins.
Principles of Time of Flight (TOF) MS
After the sample-matrix- crystals are ionized, the ions head towards the mass analyser
where they are separated according to their mass to charge ration (m/z) values. In this
project, the mass analyser utilized and coupled with MALDI, was a time of flight (TOF)
instrument with an orthogonal ion inlet. In particular, in a TOF analyser, the ions get
separated based on the time they need to reach the detector after they get accelerated from
an electric field and drift into a flight tube, which is a field free area. This procedure is
showed in the Figure 1.2.
Figure1.2 Ionization source and TOF mass analyzer (Hoffmann& Stroobant 2007).
Additionally there are some mathematical formulas which contribute to the comprehension
of how a TOF analyser works.
Ek =
𝑚𝑢2
2
= 𝑞𝑉𝑠 = 𝑧𝑒𝑉𝑠 = 𝐸𝑒𝑙
(1)
In formula (1) it is shown that the electric potential energy, Eel which is acquired by the ions
while they are in the acceleration region of the source, is converted into kinetic energy, Ek
which is identical for all the ions. Therefore, all ions move into the field free region called
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drift path, with the same initial Ek but different velocities (υ). As it is shown in formula (2),
the velocities of the ions differentiate only if they have different masses.
2𝑧𝑒𝑉𝑠 1/2
)
𝑚
u= (
(2)
Moreover, the velocity of each ion is constant until it reaches the detector; but the time (t)
an ion needs to move in the drift path until it reaches the detector is different.
𝑡=
𝐿
(3)
𝑢
As it is suggested from the name of the analyser, time formula (3) is the determining factor
for the separation of the ions of a sample (Hoffmann & Stroobant 2007).
MALDI-TOF MS is suitable for measuring compounds with molecular weight up to 200.000Da
(Caprioli, Farmer & Gile 1997); ergo it was appropriate for the MS analysis of peptides, lipids
and proteins that were used in this project.
Principles of Secondary Mass Spectrometry (SIMS)
SIMS is a mass spectrometry technique that has been used since the 1960s and it is
especially employed for high spatial resolution MSI analysis. Its principle involves a primary
ion beam, which hits the surface of a thin layered sample with 5-25kV energy and induces
the production of secondary ions from the sample. Nowadays, primary ion guns, which are
the cornerstone of a SIMS instrument utilize primary metal ions such as Au+, In+, Bi+, Xe+ and
Ga+ . After the secondary ions of the sample are induced, they are accelerated due to a high
voltage system, towards the mass analyser. One of the most regularly used mass analysers
coupled with SIMS, is a TOF analyser (Chughtai 2012). The SIMS instrument in the BIMS
laboratory is a Physical Electronics TRIFT II high resolution Secondary Ion Mass Spectrometry
TOF-MS system and it was used only once for the profiling of a small area of a sample
employed in this project, since it was too large to conduct a SIMS imaging experiment.
Compounds employed throughout the project
Sphingomyelin (SM)
Figure 1.3. SM molecular structure,Sigma-Aldrich information sheet
The sphingomyelin from chicken egg yolk used throughout this project was purchased from
Sigma-Aldrich. Sphingomyelin is a sphingolipid, that is polar and it is widely used for
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pharmaceutical purposes, cosmetics and skin care (Karlsson, Michélsen & Odham 1998).
Moreover, sphingomyelin is a compound of interest in several research studies because it is
from the (significant) components of blood and nervous tissue and is contained in plasma
membranes of higher animals.
1, 2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)
Figure 1.4. DPPC molecular structure,Sigma-Aldrich information
sheet
The DPPC, which was also purchased from Sigma-Aldrich, is a phospholipid that consists of
two palmitic acid moieties. DPPC is one of the various lipids that contain a choline group and
it is found in abundance in eukaryotic cells (Attwood, Choi & Leonenko 2013).
Angiotensin II (Asp-Arg-Val-Tyr-IIe-His-Pro-Phe)
Figure 1.5. Angiotensin molecular structure,Sigma-aldrich information
sheet
The angiotensin II was a component of a peptide
mixture prepared for the purpose of this
project and was also purchased from Sigma-Aldrich. This peptide is synthesized when the
angiotensin converting enzyme causes a cleavage in the C-terminal (-His-Leu) of angiotensin
I. Angiotensin II, plays a significant role in controlling cardiovascular structure and
hemodynamics. Last but not least, angiotensin II enhances the density of micro-vessels and
triggers angiogenesis (Product Information sheet from Sigma- Aldrich). The peptide mixture
used in this project was prepared according to a Brucker calibration standard, which is
employed for the calibration of Matrix Assisted Laser Desorption Ionization -Time of Flight
(MALDI-TOF) mass spectrometers (Instructions for use- Peptide calibration standard).
Substance p
Figure 1. 6 Sunstance P molecular structure,Sigma-aldrich information
sheet
The substance p was the second component of the already mentioned peptide mixture and
was purchased from Sigma-Aldrich too. Substance p is a peptide synthesised from 1 amino
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acid residues and it is abundant in the peripheral and central nervous system. In the former
system, it induces vasodilation, smooth muscle-contracting and hypotension while in the
latter it is believed to participate in the sensory nerve transmission (Product Information
sheet from Sigma-Aldrich).
Bovine serum albumin (BSA)
Bovine serum albumin was purchased from Sigma-Aldrich as well, and was one of the
proteins used in a protein mixture that was employed throughout this project. The proteins
for the mixture were chosen based on the Brucker Protein standard II, which is a mixture of
proteins useful for the calibrating and testing of MALDI-TOF mass spectrometers
(Instructions for Use- Protein Standard II). Therefore, since BSA and trypsinogen were the
only proteins included in the Brucker standard that were in the laboratory when this project
was initiated, they were used until its completion.
Trypsinogen
As it is already mentioned, trypsinogen was also included in the protein mixture and was a
purchased from Sigma too. Trypsinogen is a trypsin proenzyme that formulates in the bovine
pancreas and contains a 229 amino acid chain that is cross-linked by six disulphide bridges
(Product Information sheet from Sigma-Aldrich).
Gelatin
Gelatin is widely used as a thickener and stabilizer in foods, it is also a component of
pharmaceuticals, paints, adhesives, films, artificial silk, matches and the first embedding
material ever used, in 1802(Luft 1973, Product Information sheet from Sigma- Aldrich). It is a
collagen derivative that is formed when the natural triple-helix structure of collagen, breaks
into single-strand molecules. Additionally, it can readily form gels by altering its solution
temperature and due to their biocompatibility, they have numerous applications in tissue
engineering (Tabata & Ikada 1998, Product Information sheet from Sigma- Aldrich). In
particular, gelatin has already been used in the formation of polymer scaffolds in tissue
engineering (Yang, Leong, Zhaohui, Chua & Chee-Kai 2001). The gelatine utilized in this
project was purchased from Sigma-Aldrich and the solution prepared for the needs of the
project was a 10% solution.
Carboxymethyl cellulose salt (CMC)
Figure 1.7 CMC (monomer) molecular structure, product Information sheet from Sigma-Aldrich.
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CMC, which was purchased from Sigma-Aldrich, has several applications in tissue
engineering. It is an ether derivate of cellulose, which is a natural chain-shaped polymer.
Additionally, it has numerous applications in food industry as a thickener, in toothpastes,
detergents, paints and so on. In chemistry and biochemistry it has proved to be useful
because of its biocompatibility, solubility in water and low price (Jiang, Yubao, Wang,
Xuejiang, Zhang, Wen, Jiqiu, Gong & Mei 2008, Product Information sheet from SigmaAldrich). For all the above reasons, CMC was chosen as a tissue mimetic material in this
project and the CMC solution prepared was a 2% solution.
Polyvinylpyrrolidone/ Carboxymethyl cellulose (PVP/ CMC) hydrogel
According to Rosiak, Ulański , Pajewski , Yoshii & Makuuchi (2002), “hydrogels are two or
multi-component systems consisting of a three-dimensional network of polymer chain and
water that fills the space between macromolecules”. Natural and synthetic polymers have
been used in biomedical applications such as drug delivery and tissue engineering. For the
past decade in tissue engineering, hydrogels have been used as scaffold materials in order to
engineer new tissues, which could result in revolutionary applications in medicine and could
benefit many people who suffer from tissue or even organ failure. The hydrogel employed in
this project, was synthesized in the laboratory and consisted from a polymer,
polyvinylpyrrolidone (PVP) 0, 1% and CMC 1%, in two-to-eight ratio. PVP/CMC hydrogels
have been used as wound dressing because of their good biocompatibility, moisture
retention, ventilating capability and water absorption ability; consequently, such a hydrogel
was considered a choice with great potential for this project (Rosiak, Ulański, Pajewski, Yoshii
& Makuuchi 2002, Wang , Xu, Hu, Zhai , Peng , Nho , Li & Wei 2007).
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Calibration lines
The plot of calibration lines for different concentrations of the DPPC, SM, peptide mixture
and protein mixture with the tissue mimetic materials of interest, was an important first step
in this project in order to get an idea of their ideal behaviour by using MALDI- TOF MS
Imaging; and after taking the results into consideration the next step for the development of
test arrays was decided.
Sample preparation
The calibration lines which will be shown in the successive paragraphs, are demonstrating
the absolute intensities of the analytes’ protonated ions (M+H) + with regard to their
concentrations. The concentrations, on which the calibration lines were based on, were
chosen after some profiling tests at the SYNAPT and the Ultraflex III. Therefore, after
conducting three series of spotting tests and by examining the spectra that were the
outcome of this process, the concentrations that were chosen were 1,3,5,8,10μM for DPPC,
1, 2, 5, 7, 10μM for SM, 0.5, 1, 2, 3, 5μM for the peptide mixture and 0.5, 1, 2, 3, μΜ for the
protein mixture. For the plot of the calibration lines, the experimental procedure contained
the same steps for all the compounds of interest. Firstly, for the initial series of
concentrations, H2O, purchased from Sigma-Aldrich and MeOH purchased from BiosolveChemicals were used to dilute the peptides, proteins and lipids respectively. Successively,
PVP/CMC hydrogel and CMC were used because of their capability to mimic tissue behaviour
in order to make new dilution series for the DPPC, SM, peptide and protein mixture and for
the next step of the profiling process, the most suitable matrix should be chosen.
Selection of matrix
The choice of the matrix plays a significant role in the MALDI- TOF MS, as it is mentioned in
the introduction chapter and its choice may determine the outcome of an experiment. The
most commonly used matrixes are sinapinic acid (SA), 2, 5-dihrydoxy benzoic acid (DHB) and
α-cyano-4-hydroxycinnamic acid (CHCA). SA is an appropriate choice for samples with
molecular weights over 10.000Da while CHCA and DHB for masses under 10.000Da (Cohen &
Chait 1996). Therefore either DHB or CHCA would be the more suitable choices for the
peptides and lipids employed in this project. After applying both of them on the DPPC, SM,
peptide, the CHCA, purchased from Fluka, Sigma-Aldrich, formed better co-crystals with
them; and for the proteins SA, also purchased from Fluka, Sigma-Aldrich, was chosen.
SA (MW: 224.21Da)
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CHCA (MW: 189.04 Da)
Figure 2.1 Molecular structures, taken from Sigma-Aldrich product information sheets.
Matrix solution preparation
For the preparation of a matrix solution an organic solvent has to be chosen, such as
acetonitrile (ACN) or methanol (CH3OH), then an acidic organic compound, which is the
matrix itself, and trifluoacetic acid (TFA), is added. By adding TFA, there are an increased
number of protons available for ionization (Chughtai 2012)
CHCA preparation
For the preparation of a 10mg/ml matrix solution, 100 mg of CHCA were weighed and
diluted in a 10ml mixture of 70:30 H2O: ACN or CH3OH and 0.1% TFA. (TFA was purchased
from Biosolve- Chemicals).
SA preparation
For the preparation of a 20mg/ml SA solution, 200mg of SA were weighed and diluted in a
solution of 10ml 50:50 ACN: H2O and 0.1% TFA. Both matrix solutions were placed in
ultrasonic bath for 10 minutes in order to ease the dilution.
Profiling of compounds of interest
The next step of the protocol followed included the profiling of the compounds. More
specifically, a small amount (1μl) was prelevated from each concentration prepared and was
mixed with the same amount of matrix in one-to-one ratio. Successively, 0.5μl of DPPC, SM
and the peptides mixture- CHCA, was placed on different cells of a metal target plate and the
spots were allowed to dry.
Figure 2.2 Metal target plate
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When they dried, the plate was loaded in the SYNAPT and the software program Masslynx,
Waters was used firstly for the calibration of the instrument, by profiling a polyethylene
glycol (PEG) calibration standard mixed with matrix (CHCA), and then for the setting up of
the experiment. After the calibration, the mode had to be set, which was positive in the
following cases and the detector had to be at 1850V. Then a cell on the target plate was
chosen and in the end the laser was fired at 250eV all around the cell, for the same number
of laser shots (about 50 shots) for all the compounds and concentrations. From this process,
a chromatogram and a spectrum were produced, for each cell of the target plate.
Furthermore in each spectrum, it is possible to find the absolute intensity of the m/z of
interest, by choosing the appropriate setting in Masslynx. The molecular masses for the
DPPC, SM, Angiotensin II, Substance p, Trypsinogen and Albumin bovine are listed in a table
below. The peaks taken into consideration for the plotting of the calibration lines that
follow, were the intact protonated ions of the analytes, (M+H)+ where M represents the
average molecular weight and the m/z of the ions that are detected and measured by the
TOF analyser of the SYNAPT and the Ultraflex III.
Compounds
Sphingomyelin (SM)
1,2-Dipalmitoyl-sn-glycero-3phosphocholine (DPPC)
Substance p
Angiotensin II
Trypsinogen
Bovine serum albumin (BSA)
(M+H)+
703.5
734.3
1046.54
1347.73
23982
66463
Figure 2.2 Intact protonated molecules
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DPPC calibration lines
In the Figure 2.3, there is an example of a combined spectrum based on which one of the
calibration lines of DPPC was plotted.
Figure 2.3 Combined spectrum from profiling of 1, 3, 5, 8, 10μM of DPPC-CMC
6.00E+02
Absolute Intensity
5.00E+02
4.00E+02
3.00E+02
DPPC
DPPC -CMC
2.00E+02
DCCP-HYDROGEL
1.00E+02
0.00E+00
1
3
5
8
10
Concentration ( μΜ)
Figure 2.4 Calibration lines for DPPC
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In Figure 2.4, the calibration lines for DPPC are not completely linear and it was assumed
that normalization was needed in order to overcome this problem.
2.50E-01
Absolute Intensity
2.00E-01
1.50E-01
DPPC
1.00E-01
DPPC -CMC
DCCP-PVP:CMC
5.00E-02
0.00E+00
1
-5.00E-02
3
5
8
10
Concentration ( μΜ)
Figure 2.5 Normalized calibration lines for DPPC
The Figure 2.5 occurred after normalizing the values of the absolute intensities by dividing
them by the Total Ion Count (TIC) from every profiling of each concentration used for the
construction of the calibration lines. Even though normalization took place, the calibration
lines did not become linear thus it was assumed that either there was a human error in the
sample preparation, or the normalization that was conducted was not the appropriate one.
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SM calibration lines
1.40E+03
Absolute Intensity
1.20E+03
1.00E+03
8.00E+02
SM
6.00E+02
SM-CMC
SM-Hydogel
4.00E+02
2.00E+02
0.00E+00
1
2
5
7
10
Concentration (μΜ)
Figure2.6 Calibration lines for SM
In Figure 2.6 it is shown that the calibration lines are not linear, thus the next step was to
normalize the results, similarly to the case of DPPC. Based on the normalization applied, the
following plot was constructed.
5.00E-01
Absolute Intensity
4.00E-01
3.00E-01
SM
2.00E-01
SM-CMC
SM-PVP:CMC
1.00E-01
0.00E+00
1
-1.00E-01
2
5
7
10
Concentration (μΜ)
Figure 2.7 Normalized calibration lines for SM
Once again the normalized plot did not follow a linear trend; as a consequence it was
assumed again that either the normalization was not the appropriate one or that there was
an error in the sample preparation.
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Peptide mixture calibration lines
1.20E+05
Absolute Intensity
1.00E+05
8.00E+04
Angiot 2
Substance p
6.00E+04
Angiot 2-CMC
4.00E+04
Subs p- CMC
Angiot 2-hydrogel
2.00E+04
Subst p-hydrogel
0.00E+00
0.5
-2.00E+04
1
2
3
Concentration (μΜ)
Figure 2.8 Calibration lines for peptide mix.
In Figure 2.8 it is shown that the trend of the calibration lines for the peptide mixture is close
to linear but normalization with TIC was also conducted in order to check if the calibration
lines could be further optimized.
2.00E+01
Absolute intensitiy
1.50E+01
Angiot 2
1.00E+01
Subst p
Angiot 2-CMC
Subs p-CMC
5.00E+00
Angiot 2-CMC:PVP
Sub p -CMC:PVP
0.00E+00
0.5
-5.00E+00
1
2
3
Concentration (μΜ)
Figure 2.9 Normalized Calibration lines for peptide mix.
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Figure 2.9 indicates that the normalization based on the TIC optimised the initial results as
the normalized calibration lines to a small degree as they seem to follow a similar trend with
the non-normalized ones.
Protein mixture calibration lines
For the plot of the protein mixture calibration lines, the Ultraflex III was used instead of the
SYNAPT due to the fact that its detection range is too low for such large proteins as Bovine
Serum Albumin and Trypsinogen. It has not been possible to obtain a calibration line for the
proteins in the PVP/CMC hydrogel because of the low signal-to noise (S/n) ratio. For this
reason it was not feasible to detect any clear peak signals and to assign them to the proteins.
8.00E+04
7.00E+04
Absolute Intensity
6.00E+04
5.00E+04
BSA
4.00E+04
Tripsinogen
3.00E+04
Trypsinogen-CMC
2.00E+04
BSA-CMC
1.00E+04
Prot mix- Hydrogel
0.00E+00
-1.00E+04
0.5
1
2
3
-2.00E+04
Concentration (μΜ)
Figure 2.10 Calibration lines for protein mix.
In Figure 2.10 the calibration lines for the protein mixture appear to be linear except for the
one that corresponds to trypsinogen, for this reason they were not normalized.
Based on the non-normalized calibration lines, CMC seems to be a better embedding
medium than the PVP/CMC hydrogel for SM and DPPC, while the PVP/CMC appears to be a
better choice for the peptide mix because it causes less suppression of the absolute signal of
the protonated ions of interest. As far as it regards the protein mixture, both CMC and the
hydrogel supress the signal of the proteins involved, consequently they were excluded from
the main part of the project, where the rest of the compounds of interest were used in order
to develop arrays while they were “diluted” and embedded in materials that mimic tissue
behaviour.
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Because of the fact that the normalization of the calibration lines with TIC was overlooked
and was performed after the main part of the project, the normalized calibration lines were
not taken into account for the actual choice of the tissue mimetic material that was used
until the end of the project.
An alternative normalization technique
Another normalization technique that was tried out was by using Cresyl Violet (CV) as an
Internal Standard (IS). It was only employed for the compounds for SM, DPPC and the
peptide mixture that were mixed with the CMC, after it was chosen as the appropriate tissue
mimetic material for this project.
Cresyl violet preparation
For the preparation of a 10mg/ml CV solution, 100mg of CV power were weighed and
diluted in 10ml CH3OH.
Normalization of DPPC-CMC with CV
The sample preparation and the profiling of the solutions of interest for the construction of
the calibration lines similar to the one explained in par graph “sample preparation””, except
for the fact that the same amount (10μl) of CV solution was added to the each concentration
of DPPC with CMC.
5.00E+00
Absolute Intensity
4.00E+00
3.00E+00
2.00E+00
DPPC-CMC-CV
1.00E+00
0.00E+00
1
-1.00E+00
3
5
8
10
Concentration (μΜ)
Figure 2.11 DPPC-CMC with CV
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Normalization of SM-CMC with CV
1.80E+01
1.60E+01
Absolute Intensity
1.40E+01
1.20E+01
1.00E+01
8.00E+00
SM-CMC-CV
6.00E+00
4.00E+00
2.00E+00
0.00E+00
-2.00E+00
1
2
5
7
10
Concentration (μΜ)
Figure 2.12 SM-CMC with CV
Normalization of Peptide mixture-CMC with CV
1.20E+02
Absolute Intensity
1.00E+02
8.00E+01
6.00E+01
4.00E+01
Substance P-CMC-CV
2.00E+01
Angiotensin II-CMC-CV
0.00E+00
-2.00E+01
-4.00E+01
0.5
1
2
3
Concentration (μΜ)
Figure 2.13 Peptide mixture-CMC with CV
Apart from the calibration line that refers to DPPC which is close to linear, from the rest of
the normalized calibration lines it is indicated that adding CV was not a good normalization
method which could explained due to the fact that the CV solution was prepared with
CH3OH which diluted even further the concentration series, especially for DPPC and SM.
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Development of Test Arrays
For the development of the test arrays the protocol that was developed, included the
following steps. Firstly, the tissue mimetic materials that were chosen were frozen with
different shaped stamps in them, in a suitable container. Successively, the stamps were
removed and the cavities formed were filled with the analytes “diluted” in the tissue
mimetic materials and the block created was frozen again. The newly formed solid block was
dissected and after matrix application on the sections, they were loaded on the MALDI-TOF
MS instrument in order to acquire spectra that were lastly converted into images. These
steps are described in detail in the following paragraphs.
Choice of tissue mimetic materials
At first, it was thought that one tissue mimetic material would be used as an embedding
media both for the formation of a solid frozen block and as a ‘’solvent’’ of the compounds
that would be placed in the different shaped stamps of the block. Nevertheless, the choice
of an embedding material was difficult, even though it was indicated based on the
calibration lines that the CMC (2%) led to a lesser suppression of the compounds of interest
signal, compared to CMC/PVP; it was important to test the actual behaviour of CMC/PVP
too. As a result, CMC (2%) and CMC/PVP and gelatine (10%), were tested to check if they
would freeze homogeneously, if they would be able to get dissected and stay intact in the
process and also if they diffuse in such a degree that the signal of the lipids and peptides of
interest would be suppressed. Moreover, after testing PVP/CMC, gelatine and CMC as
embedding mediums in all combinations possible, for the formation of the block and the
“dilution” of the compounds, gelatine proved to be the most suitable for the block formation
and CMC for the dilutions. PVP/CMC has not been found useful because it does not freeze
homogeneously thus the sectioning of its solid block, was impossible and also in the event of
a relatively intact section, when it was desiccated, the hydrogel diffused and covered the
compounds. To conclude, after several tests CMC (2%) proved suitable as a tissue mimetic
material with which the lipids and peptides were mixed, and gelatine (10%) resulted in being
the best material for the formation of a solid block that contained the compounds in the
CMC.
Choice and design of proper container
The design and construction of a proper container for the freezing of a tissue mimetic
material that could be able to hold stamps of different shapes, was a real challenge. At first it
was considered a good idea to 3D- print a box; yet the plastic that is used in this kind of
printers was not firm and it broke very easily after it froze. Another problem emerged when
removing the frozen block from of the box and it resulted in the breaking of the plastic into
small fibber-like pieces. Consequently, the plastic box was rejected and the next idea
involved utilizing a metal one. The metal box was constructed in the workshop of AMOLF
and the stamps on the cap of the box are also metallic and they have three different shapes;
squares, circles and triangles. The stamps are connected to the cap by screws so they could
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be readily removed. In the end, this metal box worked perfectly for the formation of a block
of embedding material; however the block that came out of the box was too large for the
Cryostat, a device used for the dissection of the block. Thus, the metal cap with the shaped
stamps was put into the embedding material in a plastic ice cube tray which resulted in
being the most efficient and simple container.
Figure 3.1 Metal stamps of different shapes
Sectioning
The next step of the developed protocol included the formation of a solid block of gelatine
and its dissection. In particular, gelatine (10%) was poured into an ice cube tray with the
metal stamps inside and the tray was placed in the freezer until a solid block was created.
Then, the stamps were taken out and different shaped cavities were formed. Specifically, the
circle- cavities were filled with different concentrations of SM-CMC, the triangle- cavities
were filled with various concentrations of DPPC-CMC and in the squared- cavities the
peptide mixture-CMC was deposited. Successively, the ice cube tray was placed again in the
freezer and the result was a solid block of gelatine with the compounds of interest also
frozen in the shapes of the stamps.
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Figure 3.2 Gelatin block with developed arrays
Figure 3.3 Microtome Cryostat, Microm HM535
Moreover, after removing the gelatine block out of the ice cube tray, it was placed in a
instrument called Microtome Cryostat, Microm HM535. With Microtome, the intact sections
that were obtained were 10μm thick and the sample stage temperature was around -25OC.
Figure 3.4 Gelatin section on a glass slide.
Matrix application
The application of CHCA had to be performed, after the section was placed in the dessicator
for some time. For the homogeneous application of the matrix, a sprayer device, ImagePrep
(Bruker, Bremen, Germany) was employed at first, but because of the fact that application
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was not always homogeneous and because it is a time consuming device, another device
SunCollect (SunChrom, Friedrichsdorf, Germany) was utilized. With SunCollect, an aerosol of
matrix is applied on the sample and a homogeneous layer is formed within 10 to 30min. In
this device, the air pressure is applied is 2 bar, the distance between the sample and the
spray nozzle is manually set at 25cm, the layers of matrix are also chosen by the operator
and are between 10 and 20 and the flow of the matrix solution, starts from 5uL/min and
builds up to 30uL/min. The matrix is deposited on the section and then the organic solvent
of the CHCA solution in this case, extracts the molecules of the section and rapidly
evaporates. After the evaporation, the formation of matrix crystals occurs from the organic
acid of the solution.
MSI analysis in positive mode and Data Analysis
When the coating of the section with CHCA was homogeneous, afterwards it was loaded into
the mass spectrometer, SYNAPT in order to perform the MSI analysis. During the imaging
experiment, firstly the sample was scanned and an area of interest was selected. Then the
experiment was set up in MALDI-TOF linear mode via the Masslynx Software. In addition,
after choosing the area of interest of each sample, setting the resolution at 250x250μm and
the laser frequency at 250-300Hz, the experiment is ready to start.
Data Analysis- Biomap
After each analysis, the raw data acquired which include numerous spectra, were converted
into imaging data that are displayed with the Biomap Software (Novartis, Basel, Switzerland,
www.maldi-msi.org). In Biomap, differences at specific values in an area of interest can be
observed readily and the changing in certain pixels can be further analysed when combined
with the observing of the related spectrum. Below in Figure 3.5 there are some images at the
m/z of (M+H)+ of each compound of interest, in particular for SM: 703.5Da, for DPPC:
734.5Da, for Angiotensin: 1046.6Da and Substance p: 1347.50Da. The Figures 3.5 and 3.6
show two different sections and their difference lands on the sample preparation; in the
Figure 3.5 the CHCA application was performed with the ImagePrep device while in the
section depicted in Figure 3.6, CHCA was applied with the Suncollect device.
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SM (M+H) + m/z: 703.5
DPPC m/z (M+H) + 734.4
Angiotensin II (M+H) + m/z: 1046.6
Substance p (M+H) + m/z: 1347.5
Figure 3.5 Section 1 - CHCA applied with ImagePrep
If the sample preparation and the MSI analysis had succeed ,in the m/z values that
correspond to the (M+ H) +of each compound of interest, only one array should illuminate. In
particular, at 703.5Da, only the array of the circles should be illuminated and because in
each circle there was a different concentration of the SM-CMC mixture the intensity of the
emitted light should be different. Likewise, at 734.5Da only the triangle array with the DPPCCMC should illuminate and at 1046.5 and 1347.6Da for angiotensin II and substance p –CMC
the square-array should illuminate similarly.
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SM (M+H) + m/z: 703.5
DPPC m/z (M+H+Na) + 756.4
Angiotensin II (M+H) + m/z: 1046.6
Substance p (M+H) + m/z: 1347.5
Figure 3.6 Section 2 – CHCA applied with Suncollect
In Figure 3.6, where a second section (section 2) is depicted, it can be observed that CHCA is
more homogenously layered compared to the first section, however at the (M+ H) + m/z
values of the compounds of interest, there is no difference at the light intensity of the
arrays. The next step was to check other m/z values that correspond to (M+ H+ Na) +,
(M+H+NH4) +, (M+ H+ K) +, (M+ H+ ACN+ Na) +of each compound. Unfortunately, this step did
not bear fruits for all the compounds but only for the (M+H+ Na) + of DPPC-CMC as it shown
at the Figure 3.6.
MSI analysis in negative mode and Data analysis
It was also decided to try a change in the protocol followed, and tune the SYNAPT in
negative mode. Furthermore, in order to conduct a MALDI-TOF MS analysis in negative
mode, the matrix choice should be different thus 9-Aminoacridine (9AA) was applied in a
third section and the rest of the protocol remained the same to the one followed in the
positive mode. After acquiring the raw data of the analysis were converted into Biomap data
and led to the below images.
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SM (M-H) - m/z: 702.5
DPPC m/z (M-H) - 733.4
Angiotensin II (M-H) - m/z: 1045.6
Substance p (M-H) - m/z: 1346.5
Figure 3.7 Section 3. Biomap images- negative mode
After examining the Biomap images at the m/z values corresponding to the (M-H) - , (M+ Na H) - , (M-TFA-H)-of each compound, it was shown that neither in the negative mode the
developed arrays were able to deliver the desired results and show different light intensities
at different m/z values.
Principle Component Analysis (PCA) and Discriminant Analysis (DA)
Principle Component Analysis was also conducted as a complimentary procedure to the data
acquired from the MSI analysis. PCA is the foundation of multivariate data analysis and is an
important statistical tool for researchers. (Wold, Esbensen & Geladi 1987). According to H.
Abdi and L.J Williams (2010) “PCA is a multivariate technique that analyses a data table in
which observations are described by several inter-correlated quantitative dependent
variables”. With PCA, it is possible to acquire significant information that concerns the table
of data, to convert them into orthogonal variables which are called principal components
and present the correlation of the variables as points in histograms. Discriminant analysis
(DA) is also used in statistics in order to classify samples of interests (Poulsen & French
2004). DA in combination with PCA can offer researchers useful information that could verify
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or disprove initial indications of experiments. In this project, PCA and DA were combined,
but the information acquired was limited.
PCA and DA analysis for section2- positive mode
In order to start the PCA analysis, a conversion of the Biomap file of the section 2 into a
MATLAB file has to be performed. Subsequently, the functions ‘’Peakpicking’’ and ‘’Regions
of interest’’ (ROIs) in MATLAB were employed and each shape was considered a ROI. In
particular, in the 1, 2, 3, 4 there was SM-CMC; where in 1, the highest concentration (10μM)
of the mixture was contained. Likewise in 5, 6, 7 ROIs there was DPPC-CMC and in 8, 9, 10,
11 ROIs there was the peptide mixture. The combination of the ROIs of PCA and Discriminant
Analysis (DA) let to Figure 3.8.
1
2
4
3
5
8
9
6
7
10
11
Figure 3.8 Image of ROIs
When using the DA function, there seemed to be a classification that showed a correlation of
the peptide mixture and the CHCA and a correlation between the DPPC, SM and the CMC.
These correlations were deduced from the Figure 3.8 and its conversion into Figure 3.9.
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Peptides
Lipids
CHCA
CMC
Figure 3.9
CMC
m/ z
CHCA , Gelatin
Figure 3.10
Based on the reference spectra of the gelatine and CMC with CHCA, it is known that most of
the m/z values at the negative part of the histogram correspond to the gelatine and the
CHCA and the majority of the values at the positive part occurred due to the CMC.
Additionally, due to the PCA and DA, some histograms presented some indications of the
existence of the SM-CMC solution in the array of circles where it was supposed to be in the
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second section.
+
(M+H+Na)
m/ z
Figure 3.11 Normalized Histogram
In particular, as it is highlighted by the arrow in Figure 3.11, the peak at the m/z: 725.2Da
could have occurred due to (M+ H+ Na) +. While in the non-normalized histogram, the signalto-noise ratio (S/n) was low, as a result no conclusions could be drawn except for when it is
converted to another also non-normalized histogram-Figure 3.13, where it seems that the
ROIs 1, 2, 3, 4 are separated; presumably based on the different concentrations of the SMCMC solutions in the developed arrays. In particular, the lowest concentration which was
2μM and the highest which was 10μM appeared to separate completely, while the
concentrations 5,7μM seemed to overlap.
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m/z
Figure 3.12 Non- normalized histogram- SM
Figure 3.13 Non-normalized histogram2- SM
PCA and DA for third section- negative mode
Even though there were no certain conclusions from the PCA analysis for the second section
which was previously described, due to the fact that there were some indications about the
SM-CMC developed array, the same PCA and DA analysis took place for the third section for
which the MSI analysis was in negative mode.
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m/ z
Gelatin
Figure 3.14 Non-normalized histogram- SM
m/z :(M-H)- = 702.4
m/ z
Figure 3.15 Normalized histogram- SM
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From the PCA and DA conducted for the SM-CMC array, in negative mode, the only
indication of existence of SM was a small peak at the m/z: 702.4Da that could be assigned to
the (M-H)-, but since there were no further indications, no other histograms are to be
shown. Therefore, the peak at 702.4Da could be coincidental and belong to gelatine and not
be an SM peak.
Profiling of DPPC and SM arrays with Secondary Ionization Mass
Spectrometry (SIMS)
As it is already mentioned in the introduction chapter, SIMS was not repeatedly employed in
this project. This was the case because of the fact that the samples (sections) were too large
for this technique; despite that fact SIMS was used once in order to conduct a profiling of
the arrays developed for DPPC and SM, but not for the peptides and the protein mixtures as
the detection range of SIMS is not suitable for such compounds. For the profiling, the only
condition was that the sample should be placed on a conductive Indium Tin Oxide (ITO)
coated slide and then the slide could be placed into the TRIFT II high resolution Secondary
Ion Mass Spectrometry TOF-MS
DPPC array
Figure 3.16
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In the Figure 3.16 there is a combined spectrum and each spectrum corresponds to a
different DPPC-CMC concentration of the developed arrays, and the peak observed at m/z:
184Da could be assigned to the phosphorylcholine head group. The fact that this peak is so
small and the fact its size is almost the same to every spectrum doesn’t seem to shown any
correlation with the different concentrations. If the DPPC was actually in this area, the peak
would be much higher.
SM array
Figure 3.17
The SM has the same phosphorylcholine head group as DPPC, therefore the peak at m/z 184
could also be an indication of the existence of SM in the developed array.
Despite the observed peak at m/z 184, no certain conclusions could be drawn either for the
DPPC or for the SM, since no other adduct ions of them were observed.
Last but not least, due to a high resolution camera of SIMS, it became clear that even though
the sections from the gelatin block with the arrays of compounds seemed to be consistent,
the arrays were not homogeneous. This also led to the realization that methanol used for
the initial dilution of SM and DPPC, was responsible for the non-homogeneous freezing of
the arrays.
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Spatial resolution experiments
One of the targets of MSI is the visualization of spatial organization of biological compounds
(Jungmann & Heeren 2012) such as the lipids, peptides and proteins employed in this
project. As an extension of that, the last part of this project included a series of experiments
regarding the spatial resolution imaging, based on which, conclusions about the sensitivity of
MSI techniques such as MALDI TOF-MS and the instruments employed, can be drawn.
Sample preparation
The sample preparation included similar steps for the lipids and peptides used throughout
this project. First of all, the highest concentrations of the solutions for each compound of
interest were chosen, hence 10μΜ solution of DPPC and SM, and 5μΜ solution of peptide
mixture was used. Each compound was mixed in one-to-one ratio with a suitable matrix,
which was CHCA for the lipids and peptides and SA for the protein mixture, and they were
sprayed with Suncollect (SunChrom, Friedrichsdorf, Germany) or ImagePrep (Bruker,
Bremen, Germany) (whichever instrument was working) through a plastic mold. With this
process, each solution was sprayed on the glass and the shapes of the cavities of the plastic
mold were formed on the slide. The plastic mold was constructed by a 3D printer and it had
four arrays of different shapes; circles, triangles, squares and rectangles of different sizes.
.
Figure 4.1 3D- printed plastic mold
The last step of the sample preparation was to load the glass slides with the lipids and the
peptide mixture into the SYNAPT and set up the experiments in order to conduct the MSI
analysis. The settings of each spatial resolution experiment, included laser frequency of 250300Hz, and the resolution was set at 200μm x 200μm.
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Acquired Data from MSI analysis of DPPC
The acquired data from each experiment were converted into Biomap files and then they
were processed further with a Biomap function with which Regions of Interest (ROIs) were
selected in order to determine the exact areas with the DPPC-CHCA in mm2. Successively, a
setting in Biomap called Statistics calculated the mean intensity of the ROIs, at m/z value of
734.5Da which is the (M+H) + of DPPC, as it is already mentioned. The results of this process
let to the following images and plots.
Figure 4.2 DPPC-CHCA Biomap image
.
300.00
Mean Intensity
250.00
200.00
150.00
Mean Intensity
100.00
Normalized Intensity
50.00
1.000
1.360
1.480
1.760
1.840
1.920
2.000
2.400
1.840
2.960
2.760
3.240
3.320
3.920
4.200
4.680
4.760
0.00
Area (mm2)
Figure 4.3
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In Figure 4.3, the red line refers to the mean intensity in relation to the regions of interest in
mm2 and although it was expected for the trend of the line to be straight or constant, this
was not the case. This could be explained because of the non-homogenous spraying of the
DPPC-CHCA, or because of the manual drawing of the regions of interest. Subsequently, the
data were normalized by dividing the mean intensity of each square with the area that
corresponds to it. The normalization process is indicated with the blue line in Figure 4.3 and
it is possible to observe the better linearity of the line.
Acquired Data from MSI analysis of SM
The data were acquired similarly to DPPC and processed correspondingly with Biomap
software at the m/z: (M+H) +is 703.5Da for SM and the following image and plot were the
results of the MSI analysis.
Figure 4.3 SM-CHCA Biomap Image
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Mean Intensity
10000
9000
8000
7000
6000
5000
4000
3000
2000
1000
0
Mean Intensity
Normalized Intensity
1.8 2.08 2.12 1.6
1.2 1.28 1.32 1.2 0.88 0.84 1.32
Area ( mm2)
Figure 4.4
In Figure 4.4, the red line refers to the mean intensity in relation to the ROIs in mm2 and
even though it was expected the trend of the line to be straight or constant again, this is not
the case. This could occur due to similar errors to the DPPC in the spraying step or again due
to the manual drawing of the ROIs. Successively normalization was performed similarly to
the normalization of DPPC-CHCA. The normalization is depicted with the blue line in Figure
4.4 but there was not any significant improvement in the linearity of the trend.
Acquired Data from MSI analysis of Peptide Mixture -Substance p
After the MSI analysis, the data processing was similar to the one for DPPC and SM.
Moreover, the only difference was the m/z ratio that corresponds to the protonated ions of
Substance p which is (M+H) +: 1347.5Da.
Figure 4.4 Substance P- CHCA Biomap image
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35
Mean Intensity
30
25
20
15
Mean Intensity
Normalized Intensity
10
5
1.28
3.24
3.52
2.84
4.8
3.96
4.04
4.68
5.32
5.64
5.16
5.32
6.4
6.48
8.16
8.44
8.4
0
Area (mm2)
Figure 4.5
In Figure 4.5, the blue line refers to the mean intensity in relation to the ROIs in mm2 and
despite the fact that we would expect the trend of the line to be straight or constant, this did
not occur. This could be explained similarly to the already mentioned DPPC and SM-CHCA
due to errors in spraying of peptide mixture-CHCA onto the glass slide, because of the fact
that the mixture kept clogging the spraying head of the ImagePrep, or due to the manual
drawing of the ROIs. Furthermore, the following step was to perform normalization of the
results similarly to the normalization of DPPC-CHCA. The normalization is depicted with the
red line in Figure 4.5 and an improvement of the line linearity is observed.
Acquired Data from MSI analysis for Angiotensin II
Successively, when acquiring the data from the MSI analysis, the following step was similar
to the step followed for Substance p. Moreover, the m/z chosen in Biomap corresponds to
(M+H) +: 1046.5Da and due to the afore- mentioned process, the following image and plot
were constructed.
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Figure 4.6 Substance p-CHCA Biomap image
50
45
Mean Intensity
40
35
30
25
20
Mean Intensity
15
Normalized Intensity
10
5
1.44
3.12
3.04
4.12
4.64
4.2
5.16
4.8
5.32
6.12
6.68
6.56
7.08
7.36
8.16
8.36
8.72
0
Area (mm2)
Figure 4.7
In the Figure 4.7, the red line indicates the mean intensity in relation to the ROIs and the
trend of the lines are not again what was expected. The reasons which could be responsible
for that are already mentioned in the other peptide used in the peptide mixture. In order to
check if the results could be optimized, they were normalized and in this case, the
normalization was better than for any other compound so far, as it is shown with the blue
line of the plot, which is very close to linear.
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Protein mixture- Sample preparation
As it is already mentioned, the sample preparation of the protein mixture was different to
the other compounds to some extent. First of all, the protein mixture was not mixed with
the matrix and sprayed with ImagePrep because this mixture would clog the spray head of
the device; thus the protein mixture was firstly sprayed through the plastic mold and then
the matrix, SA was sprayed over it, onto a conductive ITO slide. Furthermore, due to the fact
that the deposit of the SA was not enough for an MSI analysis and due to lack of time, an
extra small amount (1μΜ) of SA was deposited on each sprayed on area of the ITO slide with
protein mixture; this is why some of the shapes on the image below were irregular. The MSI
analysis was conducted with the Ultraflex III, Brucker as it is more appropriate for such large
compounds as the proteins used, but the same resolution with the rest of the compounds
was set at 200μm x200μm and a linear positive mode was also set.
Acquired Data from MSI analysis of BSA
After acquiring the data from the MSI analysis of the protein mixture, the following steps
included converting the data into Biomap data, processing them further in Biomap by
drawing the ROIs and calculating the mean intensity of BSA with SA in the ROIs at m/z
(M+H)+ : 66.294 Da.
Figure 4.8 BSA Biomap image
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5
4.5
4
Mean Intensity
3.5
3
2.5
Mean Intensity
2
Normalized Intensity
1.5
1
0.5
0
Area (mm2)
Figure 4.9
In Figure 4.9 the blue line refers to the mean intensity in relation to the ROIs. Due to the
unorthodox sample preparation and the manual drawing of the ROIs, it was expected that
the blue line would not be linear or constant; hence the results should be normalized.
Nevertheless, even with the normalization, the trend of the line did not become linear but it
only got smoothened in some areas.
Acquired Data from MSI analysis of Trypsinogen
The process of acquiring and processing further the data from the MSI analysis, was
performed similarly to BSA, with the only difference lying in the m/z that corresponds to the
(M+H) + which is 22.223 Da. Based on this process, the following image and plot were
constructed.
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Figure 4.10 Trypsinogen Biomap image
6
Mean Intensity
5
4
3
Mean Intensity
2
Normalized Intensity
1
0
Area (mm2)
Figure 4.11
In the Figure 4.11, the blue line shows to the mean intensity in respect to the ROIs. Because
of the unorthodox sample preparation and the manual drawing of the ROIs, it was expected
that the blue line would not be linear or constant; consequently, the results should be
normalized. Despite the normalization conducted, the trend of the line did not become
linear as it can be observed with the red calibration line.
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Results, discussion and suggestions
There were several conclusions drawn from this research project. First of all, the initial idea
to employ a non-commercial hydrogel prepared from PVP/CMC as a tissue mimetic material,
proved to be a non- efficient choice for this project as it suppresses the absolute intensity of
the intact, protonated and other adduct ions of DCCP, SM and peptide mixture. Moreover it
completely suppresses the signals of the protein mixture. PVP/CMC was also considered
unsuitable for this project, due to its difficulty to be sectioned. Taking the above into
account and after many sets of trial experiments, CMC and gelatin proved to be better tissue
mimetic materials. Even though they met our expectations, the results about the developed
arrays from the MSI were not satisfactory, due to the fact that the developed arrays were
not distinguished just by observing an image created at specific m/z values. Nearly at the
end of the project, it was realized that the developed arrays did not behave as expected,
because of the fact that the DPPC and SM were initially dissolved in methanol and then for
the creation of the concentration series- arrays they were dissolved in CMC. In particular,
the high intensity of water in CMC prevented its mixing with methanol, thus the lipids-CMC
solutions had two phases and even though they seemed homogeneous after vortex, when
the solutions were placed into the cavities of the gelatine to form a frozen solid bock, the
freezing was not homogeneous either. In addition, the fact that the freezing point of MeOH
is around -100oC, made its freezing impossible.
Figure 5.1 Two phases in DPPC and SM with CMC
solutions.
All the above led to the conclusion that this project could not be employed as a
quantification method for MSI techniques and instruments involved in it.
As far as it regards the spatial resolution experiments, from the MSI analysis and the plots
constructed for DPPC, SM, peptide and protein mixture mixed with CHCA and SA
respectively, it became clear that the Suncollect is a better device than ImagePrep for the
sample preparation in such experiments. It was also indicated that the correlation between
the intensity of the solutions at the (M+H) + values of each compound of interest and the
ROIs was not completely linear but it could probably be, if the human error was reduced and
if the Suncollect was employed for all the experiments. Last but not least because of the
Eleni Skandalaki
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Page 45
spatial resolution experiments, it was indicated that the MALDI-TOF instruments used in the
BIMS laboratory are of high sensitivity and their limit of detection is also high.
Suggestions
In the event of a repetition of this project a suggestion would be to employ a commercial
hydrogel as a tissue mimetic material. Moreover for the plot of the calibration lines of the
biological compounds of interest it would be more accurate to use more adduct ions and not
just their protonated ions. As far as it concerns a normalization method for the calibration
lines, a good idea would be to employ deuterium (2H) as an internal standard. On the other
hand, if the biological compounds chosen for a future project are diluted in a solvent that
actually mixes with the tissue mimetic material, then maybe the normalization methods
would not be necessary.
Eleni Skandalaki
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Appendix
The combined spectra base on which the calibration lines where constructed
DPPC
Figure 6.1 Combined spectrum of DPPC
Figure 6.2 Combined spectrum of DPPC- PVP/CMC
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SM
Figure 6.3 Combined spectrum of SM
Figure 6.4 Combined spectrum of SM-CMC
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Figure 6.5 Combined spectrum of SM-PVP/CMC
Peptide mixture
Figure 6.6 Combined spectrum of Peptide mixture
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Figure 6.7 Combined spectrum of Peptide mixture-CMC
Figure 6.8 Combined spectrum of Peptide mixture- PVP/CMC
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Intens. [a.u.]
Protein Mixture
x104
6
4
2
0
30000
40000
50000
60000
70000
80000
90000
100000
m /z
Intens. [a.u.]
Figure 6.10 Combined spectrum of Protein Mixture
2000
1500
1000
500
30000
40000
50000
60000
70000
80000
90000
100000
m /z
Figure 6.11 Combined spectrum of Protein mixture- CMC
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Intens. [a.u.]
175
150
125
100
75
50
20000
25000
30000
35000
40000
45000
50000
55000
60000
65000
70000
m /z
Figure 6.12 Combined spectrum of Protein mixture- PVP/CMC
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