Tuning the Selectivity of MALDI Imaging Mass Spectrometry through Control of the Sample
Preparation Parameters of Alternative Matrix Deposition Techniques
Brian Malys, Elsa Gorre, Kevin Owens
Department of Chemistry, Drexel University, Philadelphia, PA 19104
Introduction
Results
Results
In MALDI mass spectrometry the peaks observed in the resulting mass spectrum is dependent on the details of
sample preparation. These details include the selection of the matrix, solvents, deposition method, and matrix-toanalyte ratio, among others. These choices affect the selectivity of the experiment, resulting in biases toward certain
analytes. In a MALDI imaging experiment, there can be hundreds of analytes belonging to different classes of
compounds and the biases introduced during sample preparation can result in a number of analytes of interest not
being detected [1]. Due to the clear utility of MALDI imaging on complex biological samples, a better understanding of
the effect of sample preparation parameters on the selectivity of a MALDI imaging experiment is of interest.
Electrospray deposition (ESD) allows for a homogenous and reproducible application of matrix to a sample, making it
well suited for these studies.
Effect of the Choice of the Matrix
Effect of Sampling Parameters
In Figure 6, four matrix/cationization agent systems were sprayed on identically prepared sets of analytes. While
some heterogeneity is expected from sample to sample due to the application of analyte by the dry drop technique, there
are several combinations of analytes and matrix systems which do not produce signal. Neurotensin is not detected when
either dithranol system is used, polystyrene is detected only in the dithranol and AgTFA system (as Ag+ is required for
cationization), and the polyethylene glycol is detected over a much larger area when dithranol was applied as compared
to CHCA.
Intens. [a.u.]
a
2352.782
bradykinin
200
3185.687
2040.468
3289.792
3497.881
Intens. [a.u.]
0
b
125
2154.366
1890.164
2198.380
1846.133
100
2242.429
1802.081
1758.063
75
2286.450
1713.994
50
2330.455
1669.960
1625.942
25
Intens. [a.u.]
2014_04_02 4analytes 4martixsystems 0:R00X162Y100 MS
2022.284
2374.455
2418.524
0
2014_05_07 CHCA and SA with pep mix 0:R01X183Y338 MS
1537.352
c
300
200
1619.418
1904.862
1060.897
100
1639.222
2128.767
0
1000
1500
2000
2500
b
c
matrix
gramicidin
bradykinin
fibrinopeptide A
gramicidin
Figure 7. Locations of analytes applied by dry drop and
approximate area of matrix applied (the large green
circle) by ESD for images shown in figures 8 and 9.
Figure 8. MALDI images of a single sample with
different mass filters applied.. The mass filters are (a
and d) 1061 +/- 2.6 Da for bradykinin, (b and e) 1537
+/- 3.8 Da for fibrinopeptide A, and (c and f) 1903 +/4.8 Da for gramicidin. Four analyte solutions were
applied as 1 uL of solution in 90/10 methanol/water by
the dry drop technique. The solutions applied were
0.33 mg/mL of bradykinin in water, 0.33 mg/mL of
fibrinopeptide A in water, 0.33 mg/mL of gramicidin in
water and 0.33 mg/mL of each analyte in methanol as
pictured in Figure 7. 0.01 M CHCA in methanol was
applied by ESD from a spray height of 2 cm, a flow rate
of 5 μL/min, an applied voltage of 4.8 kV, and a spray
time of 45 s. The color scale has been adjusted
between the top and bottom set of images.
3081.587
1936.294
150
a
3000
3500
Figure 2. Representative spectra of (a) polystyrene 2430 in dithranol with
AgTFA, (b) polyethylene glycol 2000 in dithranol with AgTFA, and (c) a
mixture of bradykinin, fibrinopeptide A and gramicidin in sinapinic acid. For
each sample, analyte was applied by dry drop and the matrix (and
cationization agent, if required) was applied separately by ESD. Additional
detailed information about sample preparation is located in the caption for
Figure 4 for spectra a and b and in Figure 6 for spectrum c.
Figure 3. MALDI Images of bradykinin stamped from 0.25 mg/mL solution
in 90/10 methanol/water as the letter “g” and 0.01 M CHCA in methanol
solution applied separately by ESD from a spray height of 2 cm, a flow rate
of 5 μL/min and an applied voltage of 5.58 kV. The spray times were (a) 5
s, (b) 10 s, (c) 20 s, (d) 30 s, (e) 40 s, and (f) 60 s. The mass displayed is
1061 +/- 2.6 Da and the image is TIC normalized.
d
e
f
500
0
00
100
200
300
400
500
600
700
800
900
Total Laser Shots
Signal Profile of Bradykinin in DHB
40000
35000
30000
25000
20000
15000
10000
5000
0
00
100
200
300
400
500
600
700
800
900
Total Laser Shots
Figure 11. Accumulated peak area verses number of laser
pulses for sample consisting of gramicidin and DHB prepared
identically as that of Figure 10.
1000
900
800
700
600
500
400
300
200
100
0
00
In Figure 8, each of the three analytes is detected when it is spotted individually. When the three analytes are spotted
together as in the sample well on the lower right of the sample, the three analytes are not detected together. While the
signals of bradykinin and fibrinopeptide A are not greatly impacted, gramicidin is not detected when the other analytes
are present. This indicates that in this experiment, the signal of gramicidin is not only dependant on the distribution of
gramicidin in the sample, but also on the presence of bradykinin and/or fibrinopeptide due to analyte suppression.
Separate experiments (not shown) using dry drop samples indicate that it is bradykinin, and not fibrinopeptide A that
suppresses the gramicidin signal.
a
Figure 5 MALDI images of 1 μL dried drop spot of a 1 mg/mL solution of
polystyrene 2430 in THF and 0.044 M dithranol and 1.5 mM AgTFA in TFA
applied by ESD from a spray height of 1 cm, a flow rate of 5 μL/min, an
applied voltage of 5.34 kV, and a spray time of 30 s. Analyte and matrix
were applied separately in four locations, indicated approximately by the
yellow circles. The mass range displayed is 2456 +/- 12.3 Da and the
image is TIC normalized.
The effect of matrix spray time is investigated in Figures 3 and 4. As spray time increases, the amount of matrix
increases and the time in which the analyte can interact with the matrix and cationization reagent within the droplet
increases. The two locations sprayed for 30 seconds in Figure 4 and all locations in Figure 5 were prepared
identically with the exception of the flow rate used in the ESD process. Due to the diameter of the ESD spray being
approximately 0.5 cm when THF is used as the solvent, four samples could be applied to a single Multiprobe plate.
The lower flow rate used for the sample in Figure 5 would result in less total matrix being deposited on the sample.
The change in the flow rate is also expected to affect the properties of the spray and the resulting solid sample [3].
Note that higher ESD flow rates produce larger droplets containing more solvent than smaller droplets produced at
the lower flow rate. The “wetter” particles impacting the surface using the higher flow rate are expected to produce a
solid sample with better incorporation of the analyte into the matrix solid.
d
b
e
2500
2000
1500
1000
500
0
100
200
300
400
500
600
700
200
300
400
500
600
700
800
900
Total Laser Shots
Figure 12. Accumulated peak area verses number of laser
pulses for sample consisting of bradykinin and DHB prepared
identically as that of Figure 10.
Signal Profile of Bradykinin in Sinapinic Acid
3000
00
100
800
Total Laser Shots
900
700
600
500
400
300
200
100
0
00
100
200
300
400
500
600
700
800
900
Total Laser Shots
Figure 14. Accumulated peak area verses number of laser
pulses for sample consisting of bradykinin and sinapinic acid
prepared identically as that of Figure 10.
Figures 10 to 14 demonstrate how the TOFMS sampling parameters used in a MALDI imaging experiment
influence the results. Laser power was kept constant for each matrix, but varied as the matrix was changed. In three
of the samples, the early laser shots produce no analyte signal. In figures 10 and 12, signal is immediately seen.
Depending on which range of laser shots is selected to create an image, very different images would be observed.
Conclusion
Figure 9. Six images of one sample using sinapinic acid as the matrix.
Figure 4. MALDI images of 1 μL dried drop spot of a 1 mg/mL solution of
polystyrene 2430 in THF and 0.044 M dithranol and 1.5 mM AgTFA in TFA
applied by ESD from a spray height of 1 cm, a flow rate of 10 μL/min, and
an applied voltage of 5.34 kV. Analyte and matrix were applied separately
in four locations, indicated approximately by the yellow circles. The spray
times were: (a) 15 s, (b) 30 s, (c) 45 s, and (d) 30 s. The mass range
displayed is 2456 +/- 12.3 Da and the image is TIC normalized.
1000
Figure 13. Accumulated peak area verses number of laser
pulses for sample consisting of gramicidin and sinapinic acid
prepared identically as that of Figure 10.
2014_04_02 4analytes 4martixsystems 0:R00X131Y093 MS
2977.499
2144.572
100
fibrinopeptide A
2769.259
2873.390
2248.655
1500
Cummulative Peak Area
Figure 8. Six images of one sample using CHCA as the matrix.
Figure 1. Photograph of a Bruker
Multiprobe SS sample plate used
in the imaging experiments and
rubber stamps used to apply
matrix in the shape of letters.
Effect of Spraying Parameters
300
2000
Signal Profile of Gramicidin in Sinapinic Acid
Results
2456.907
2500
Figure 10. Plot of the accumulated peak area verses number of
laser pulses for a sample consisting of 0.1 mg/mL bradykinin in
90/10 methanol/water applied by ESD from a spray height of 2.5
cm, spray time of 60 s, flow rate of 5 μl/min, and applied voltage
of 5.70 kV followed by 0.10 M CHCA in methanol applied by ESD
from a spray height of 1 cm, spray time of 3 min, flow rate of 5
μl/min, and applied voltage of 5.70 kV. No laser rastering was
used.
Signal Profile of Gramicidin in DHB
Cummulative Peak Area
Samples were prepared in two steps: the application of the analyte followed by
the application of the matrix (together as a mixture with a cationization agent, when
necessary). For samples with analytes applied by stamping, a rubber stamp, shown
in Figure 1, was dipped into the analyte dissolved in a 90/10 v:v methanol/water
solution. The stamp was applied to a Kimwipe once to remove excess solution, then
pressed on the target plate. Analyte was also applied by the dry drop technique and
by ESD for other samples. For all samples, the analyte was applied first and allowed
to dry completely, before the matrix was applied by ESD [2,3]. All analytes, matrices,
cationization reagents and solvents were used as received.
All spectra and images were obtained with a Bruker (Berlin, Germany) Autoflex III
MALDI TOFMS running FlexControl (ver. 3.4). Spectra were analyzed with
FlexAnalysis (ver. 3.4). Images were collected with FlexImaging (ver 4.0). All spectra
were collected in linear mode using a Smartbeam II laser with a repetition rate of 100 Hz.
3000
Cummulative Peak Area
Methods
Cummulative Peak Area
Figure 6. MALDI images of four different samples (b-e) consisting
of four analytes applied individually as 1 μL of 1 mg/mL solution
using the dry drop technique. The analytes were polystyrene 2430
(orange, 2769 Da +/- 0.25%), β-cyclodextrin (red, 1159 Da +/0.25%), neurotensin (green, 1676 Da +/- 0.25%), and polyethylene
glycol 2000 (yellow, 1978 Da +/- 0.25%) applied as shown in (a).
Note that the polystyrene and polyethylene glycol solution spread
outside of the sample well during the application. Matrix and
cationization solution was applied by ESD with a spray height of 1
cm, a flow rate of 10 μL/min, an applied voltage of 5.34 kV, a spray
time of 30 s, a matrix concentration of 0.05 M and a cationization
reagent (if present) of 1.4 mM. The solutions applied were: (b)
CHCA in methanol, (c) CHCA and NaTFA in methanol, (d) dithranol
and NaTFA in THF, and (e) dithranol and AgTFA in THF.
Cummulative Peak Area
Signal Profile of Bradykinin in CHCA
c
f
Figure 9 MALDI images of a single sample with
different mass filters applied. The mass filters are (a
and d) 1061 +/- 2.6 Da for bradykinin, (b and e) 1537
+/- 3.8 Da for fibrinopeptide A, and (c and f) 1903 +/4.8 Da for gramicidin. Four analyte solutions were
applied as 1 uL of solution in 90/10 methanol/water by
dry drop. The solutions applied were 0.33 mg/mL of
bradykinin in water, 0.33 mg/mL of fibrinopeptide A in
water, 0.33 mg/mL of gramicidin and 0.33 mg/mL of
each analyte as pictured in Figure 7. 0.01 M sinapinic
acid in methanol was applied by ESD with a spray
height of 2 cm, a flow rate of 5 μL/min, an applied
voltage of 4.8 kV, and a spray time of 45 s. The color
scale has been adjusted between the top and bottom
set of images.
The data presented here demonstrate the dependence of a MALDI imaging experiment on many sample
preparation and sampling parameters which interact in complex ways. The spray time used will determine the
amount of matrix solution being deposited, which dictates the ratios between matrix, analytes and cationization
reagent (if present). The spray time also determines the time in which analyte can interact with deposited droplets of
matrix solution. Longer spray times may allow for better incorporation of the analyte within the matrix, resulting in
increased signal.
The composition, size and morphology of the particles/droplets being deposited by ESD vary with the ESD
parameters used. These parameters include the solvent and matrix identity, matrix concentration, flow rate and spray
height [3]. By controlling the properties of the particles/droplets that are deposited, the incorporation of analyte in the
matrix particles can also be controlled. The incorporation of the analyte would also be dependant on the
hydrophobicity of both the analyte and the matrix as well as that of the solvent. Additionally, the incorporation of the
analyte may vary within the solid sample. This would result in the selectivity of an image also being dependant on the
sampling parameters used which would include laser power and which laser shots (i.e., shots 0 to 100 or shots 100200 at a single laser location) are used to produce an image.
Even if each analyte is well incorporated, an imaging experiment may not reflect the actual distribution of each
analyte. If present, analyte suppression or enhancement effects would distort the resulting image since the image of
a given analyte would be dependant not only on the distribution of that analyte, but also on the distribution of one or
more suppressing or enhancing analytes also present in the sample. These effects also depend on the matrix
identity. The selection of the matrix and cationization reagent also impacts the selectivity of an imaging experiment.
Selection of an acidic matrix would improve the ionization efficiency of basic analytes and vice versa [5]. Analytes
with an affinity for particular cations would have different signals as the identity, amount, and distribution of the
cationization reagent was varied. This is expected to be of importance in imaging experiments of biological tissue due
to the prevalence of sodium and potassium in these samples.
Continuing work will use factorial designs to understand how the spray parameters of ESD affect the resulting
image. A wider range of analytes and matrices will be studied to determine the effect of properties such as acidity and
hydrophobicity on the selectivity of imaging experiments. Additionally, the analyte can be applied to the surface by
ESD to produce a more reproducible pattern than those made here with the dry drop technique.
References
Figure 9 show the same system as Figure 8, except that sinapinic acid is used as the matrix. Comparing the two sets
of images, there is less suppression seen when sinapinic acid is used. While there is a drop in signal when the three
analytes are spotted together, they are all detected. Gramicidin is more hydrophobic than fibrinopeptide A and bradykinin
[4] and is suppressed less when sinapinic acid, a less polar matrix, is used as compared to CHCA, a more polar matrix.
This system indicates the selectivity of a matrix is dependant not only on the interaction of a matrix with an individual
analyte, but the interaction of a matrix with the full collection of analytes.
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in polymer analysis, Ph.D. thesis, Drexel University, 2012.
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chromatography. J. Chromatogr. 359, 499-517.
5. Mahan, A., Exploration of the Quantitative Aspects of the MALDI TOFMS Analysis of Peptides, Ph.D. thesis, Drexel University, 2012.