Biology Reference
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extraneous salt adduct MS peaks due to the thorough desalting
performed before sample elution. This method provides good
peptide recovery (50-70%), and it concentrates peptides in a
small solvent volume providing signifi cantly improved sensitiv-
ity of MS analysis.
9.
There are numerous reports of additional spotting techniques,
MALDI matrices, and additives that improve the limit of detec-
tion under specifi c MALDI-MS conditions. For example, in a
modifi cation of the common dried-droplet technique, 0.5-1
L
of sample solution is deposited on the MALDI plate and then
mixed by aspiration with a comparable volume of matrix solu-
tion before drying the droplet in the air or nitrogen (
μ
15
). Since
this technique decouples sample deposition from the matrix
addition step, additional sample preparation can be performed
directly on the MALDI plate, including on-plate enzymatic
digestion and MALDI-MS calibrant addition. Dried sample
spots can be desalted directly on the MALDI plate using a
crystal washing procedure that might improve MALDI-MS
spectra by reducing extraneous adducts and ionization suppres-
sion. Vacuum drying has been reported to produce uniform
crystals in a dried-droplet method (
16
). Application of matrix
additives, e.g., ammonium monobasic phosphate or ammonium
dibasic citrate, reduces sodium and potassium metal ion cluster
interferences, thus improving peptide ion intensity and signal-
to-noise ratio for peptide samples spotted at low femtomole
level (
17
). A thin-layer spotting technique is applied to achieve
enhanced sensitivity of peptide detection, for instance, for
sample concentrations around 0.1 pmol/
L or lower (
18, 19
).
The technique requires a polished MALDI plate without
etched sample miniwells. Spotting and drying of the matrix
and sample solutions are performed separately. Because the
matrix and sample layers are thin, the number of laser shots per
desorption spot needs to be reduced to below 20. In an opti-
mized thin-layer spotting protocol, the CHCA matrix (20 g/L)
and nitrocellulose (5 g/L) are introduced as a thin layer onto
a MALDI target in acetone/2-propanol (1:1; v:v). An equiva-
lent sample volume is introduced onto the matrix surface, the
solvent evaporated, and the sample is desalted by surface wash-
ing using a small amount of water (
19
). Peptides stored on a
spotted MALDI plate are, in general, relatively stable and can
be reanalyzed days or weeks later, especially if the plate is kept
in a dry and oxygen-free environment. Tryptophan and methi-
onine are, of all amino acid residues, the most prone to oxida-
tion which may occur during MALDI-MS sample preparation.
Therefore, in order to increase protein coverage, appropriate
amino acid residue modifi cations can be included in the search
parameters during software-assisted database searches of the
μ
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