Biology Reference
In-Depth Information
Only recently, MS-based imaging techniques have been applied
for the analysis of plant samples [
9
-
12
].
Here, we present our current protocols for detecting (a) small
molecules (b) proteins and peptides in cryo-dissected immature
barley grains (a, b) and tobacco roots (a). We will describe the criti-
cal steps in detail allowing to reproduce the experiments and to
guide the set-up of MALDI imaging for other tissues. The stan-
dard workfl ow for MALDI mass spectrometry-based imaging of
molecular compounds in biological tissues is schematized in Fig.
1
.
2
Materials for MSI of Small Molecules
1. Cryostat (we are using a Leica CM3050, Leica Microsystems
GmbH Wetzlar, Germany), brush, forceps, razor blade.
2. Liquid nitrogen, optimal cutting temperature (OCT) medium,
water.
3. Indium tin oxide (ITO) glass slides (Bruker Daltonics GmbH,
Bremen, Germany).
4. Desiccator and vacuum pump.
5. Whiteout solution (Laco-offi ce products Finke GmbH,
Sottrum, Germany).
6. Stereomicroscope (e.g., Leica MZ6,) connected to a digital
camera (AxioCam ICc1, Zeiss, Jena, Germany) or a similar
microscopic image acquisition system (
see
Note 1
).
2.1 Materials for
Sample Preparation,
Cryo-sectioning,
and Slide Preparation
2.2 Materials for
Matrix Application of
Small Molecules MSI
1. ImagePrep device (Bruker Daltonics GmbH, Bremen,
Germany;
see
Note 2
).
2. Dilutions of particular matrices were prepared according to the
instructions for the ImagePrep. 2,5-Dihydroxybenzoic acid
(DHB) was diluted to 30 mg/ml in 50 % (v/v) methanol with
0.2 % (v/v) trifl uoroacetic acid (TFA), and
-cyano-4-
hydroxycinnamic acid (HCCA) to 7 mg/ml in 60 % (v/v) ace-
tonitrile with 0.2 % (v/v) TFA.
α
1. fl exControl (Bruker Daltonics GmbH, Bremen, Germany).
2. fl exImaging (Bruker Daltonics GmbH, Bremen, Germany).
3. MTP Slide-Adapter II (Bruker Daltonics GmbH, Bremen,
Germany).
4. 1:1 Mixture of PEG200 and PEG600, diluted to 1:300 in
TA30 solution (30 %, v/v, acetonitril, 0.1 %, v/v, TFA).
2.3 Software
and Materials for
MALDI-MS Image
Acquisition
1. fl exImaging (Bruker Daltonics GmbH, Bremen, Germany).
2. ClinProTools (Bruker Daltonics GmbH, Bremen, Germany).
2.4 Software for
MALDI-MS Imaging
Data Analysis
