Biology Reference
In-Depth Information
extract (black) with DHB as matrix compound compared to
the background spectrum derived from DHB alone (red). This
matrix produces very intense background signals in the
m/z
range 100-600 in positive ionization mode (Fig.
3
). Therefore,
peak selection has to be carried out manually.
9. The number of exported single spectra for analysis by
ClinProTools (CPT) has to be reduced to ca. 2,000 if no data
reduction in CPT is performed. High rate of data reduction is
associated in loss of peak resolution and has therefore to be
adapted to the number of spectra and to the kind of analysis.
Another possibility for reduction of data amount is to reduce
mass range to targeted mass signals.
10. The analysis of peptides and proteins is usually hampered by
the presence of other analytes, mainly abundant membrane
phospholipids, in the tissue. These lower mass lipids are highly
abundant and typically ionize readily. Due to competition for
ionization, the presence of lipids and other small molecules
usually suppresses the ionization of larger molecules, such as
proteins. The ethanol washes suggested here remove most of
the lipids and other small molecules, enabling the detection of
proteins. Both the solvents and the duration of the washing
steps can be modifi ed to optimize for a particular workfl ow.
For example, 2 min immersion as described above is useful
before a tryptic digest of the tissue, as it will remove endoge-
nous peptides which could otherwise interfere with the ioniza-
tion of tryptic peptides. Other, more stringent washing
procedures have been suggested, such as the inclusion of
isopropyl alcohol or chloroform [
13
,
14
]. Shorter washing
steps potentially allow detection of smaller peptides, at the
expense of the detection of higher mass proteins. Typically,
detection of proteins is limited to masses of 20-30 kDa due to
similar ion suppression effects, but specifi c washing procedures
have been suggested to allow detection of proteins up to
70 kDa [
15
].
11. Similar to the matrix deposition step, the size of the trypsin
droplets deposited on the tissue surface has direct impact on
the possible spatial resolution of the resulting peptide images.
Small trypsin droplets limit the delocalization of the peptides
but also negatively affect the effi ciency of the enzymatic diges-
tion. The ImagePrep protocol is designed to deposit the total
amount of enzyme in a series of 10-20 short (~1 s) spray bursts
followed by immediate drying with nitrogen. The setup proce-
dure described will ensure that the instrument is set up cor-
rectly to achieve a total of ~20 s spray time with a volume of
200
l. Larger volumes of trypsin (at lower concentration) are
not recommended, as the ammonium bicarbonate buffer has
negative effects on the tissue integrity.
μ
