Biology Reference
In-Depth Information
Place a glass coverslip on the ITO slide, directly covering
the senor but not the sample.
3. Fill an appropriate amount of matrix (7-8 ml) solution into the
reservoir of the spraying device. Either DHB or HCCA matrix
can be used. To compensate for the buffer capacity of the
ammonium bicarbonate in the trypsin solution, the TFA con-
tent of any matrix used should be increased to 1 % (v/v).
4. Apply matrix solutions via sensor-controlled vibrational vapor-
ization, utilizing the ImagePrep device (Bruker Daltonics
GmbH) as per the manufacturer's instructions (
see
Note 6
).
Different spray protocols specifi c for the particular matrices are
provided.
5. Immediately after matrix application clean the spray device
with methanol.
5.4 MALDI-MSI
Measurement of
Tryptic Peptides
Methods are described for Bruker ultrafl eXtreme instrumentation
(Bruker Daltonics GmbH), but are generally applicable for Bruker
MALDI-TOF instruments.
1. Fit the freshly prepared slides into a Slide Adapter II MALDI
target and introduce the adapter into the source of the MS
instrument.
2. Select the required fl exControl method. For tryptic peptides,
use refl ector mode in positive polarity and an
m/z
range of
500-4,000, and a sampling rate of 0.5 Gs/s (corresponding to
20,000-25,000 data points per spectrum). Make sure there is
enough space on the hard disk.
3. Perform mass calibration using a peptide standard (e.g.,
Peptide Calibration Standard II, Bruker Daltonics GmbH),
mixed 1:1 with the same matrix used for sample preparation
deposited next to your sample on the slide.
4. Adjust laser power and number of laser shots needed to a rela-
tive signal intensity of 25,000 by checking signal intensity from
various regions of the tissue section (
see
Note 12
).
5. Create a fi le for MSI measurements using fl exImaging software
(Bruker Daltonics GmbH). Here, the optical image of the
sample (Subheading
4.1
,
step 6
) is needed to align sample
stage position and laser spot focus of the instrument. Designate
the measurement region and select the desired spatial resolu-
tion. Typically, the spatial resolution of tryptic digests is lim-
ited to raster widths of 50
m or higher. The autoXecute
method chosen defi nes i.e., number of laser shots and the fl ex-
Control method (
see
Note 13
).
6. Perform MSI measurement. Carry out the measurement of the
spots in random order to eliminate infl uence of measurement
order.
μ
