Biology Reference
In-Depth Information
5.5 Matrix
Deposition for the
Analysis of Intact
Proteins
1. Fill the matrix vial with pure methanol and set the global spray
power adjustment for the ImagePrep as described in the manual
2. Insert the slide with digested sample into the ImagePrep
instrument and adjust slide position on the small elevation in
the way that the light sensor isn't covered by a section. 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 SA or HCCA matrix is
recommended for the analysis of intact proteins (
see
Note 14
).
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.
5.6 MALDI-MSI
Measurement
of Intact Proteins
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 slide 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 intact proteins,
use linear mode in positive polarity and a mass range of 2,000-
25,000 Da with a sampling rate of 0.1 Gs/s (corresponding to
ca. 10,000 data points per spectrum). Make sure there is
enough space on the hard disk.
3. Perform mass calibration using a protein standard (e.g., Protein
Calibration Standard I, 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.
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
) is needed to align sample stage posi-
tion and laser spot focus of the instrument. Designate the mea-
surement region and select desired spatial resolution. The
autoXecute method chosen defi nes, i.e., number of laser shots
and the fl exControl method. Additionally, mark a small mea-
surement region with just matrix as blank of approximately
25-35 laser raster points.
