Biology Reference
In-Depth Information
3
Materials for MSI of Proteins/Peptides
Sample preparation is performed in the same way as stated in
Subheading
5.1
using the same material as indicated in
Subheading
2.1
.
3.1 Materials for
Sample Preparation,
Cryo-sectioning,
and Slide Preparation
3.2 Materials for
Matrix Application of
Protein/Peptide MSI
1. ImagePrep device (Bruker Daltonics GmbH;
see
Note 2
).
2. Dilutions of particular matrices were prepared according to the
instructions for the ImagePrep. Sinapinic acid (SA) was diluted
to 10 mg/ml in 60 % (v/v) acetonitrile with 0.2 % (v/v) tri-
fl uoroacetic acid (TFA).
-Cyano-4-hydroxycinnamic acid
(HCCA) to 7 mg/ml in 60 % (v/v) acetonitrile with 0.2 %
(v/v) TFA.
α
MS image acquisition is performed in the same way as stated in
Subheadings
5.4
(peptides) and
5.6
(proteins) using the same soft-
ware packages as indicated in Subheading
2.3
.
3.3 Software for
MALDI-MS Image
Acquisition
3.4 Software for
MALDI-MS Imaging
Data Analysis
Data analysis is performed in the same way as stated in
Subheading
4.4
using the same software packages as indicated in
Subheading
2.4
.
4
Methods for MALDI-MSI of Small Molecules
1. Clean the sectioning blade or put a new one into the cryostat
and adjust the cutting temperature. For most samples, the
temperature is set to −20 °C. If the non-embedded part of the
sample tends to be still fl exible during the cutting process,
lower temperatures should be used. Otherwise, for highly frag-
ile samples test higher temperatures (i.e., −10 °C) (
see
Note 3
).
2. Fresh plant material has to be frozen in liquid nitrogen.
Transfer the frozen sample into the cryostat chamber cooled to
the desired temperature (−20 °C) and fi x it onto the sample
plate via either a droplet of water (ice) or of OCT medium
(
see
Note 4
). If necessary, precut the plant material with a
razor blade to a suitable length before fi xation. Select a proper
orientation for longitudinal or cross sections.
3. Start the sectioning process by moving the cutting block with the
sample plate. To trim the tissue sample a large section thickness
can be selected until the desired morphological parts are visible.
Then change the setting to the desired thickness. Cut 20-30
4.1 Sample
Preparation, Cryo-
sectioning, and Slide
Preparation
μ
m
sections for seeds and 35-45
m for tobacco roots (
see
Note 5
).
Transfer the fi ne sections immediately onto indium tin oxide
μ
