Biology Reference
In-Depth Information
extraction. The peptides are desalted and eluted in approximately
0.7-1.5
-cyano-4-hydroxycinnamic acid (CHCA) MALDI
matrix dissolved in a 0.1% TFA solution of a 4:1 (v:v) mixture
of ACN and water, which is suffi cient to create three MALDI
spots per sample using the dried-droplet technique. The fi rst
two spots are used directly for MALDI-MS and data-dependent
MS/MS analysis, and the remaining spot is saved as a reserve
for mass-targeted MALDI MS/MS. In the ZipTip elution
dried-droplet technique, the sample is eluted and mixed with
MALDI matrix by repeatedly drawing and expelling (ca. 20
times) the smallest possible amount of the eluting solution into
a microcentrifuge tube while avoiding passing air bubbles
through the ZipTip. MALDI matrix is dissolved in a solution
with an increased acetonitrile content (0.1% TFA solution of a
4:1 (v:v) mixture of ACN and water) with respect to the fi nal
1:1 ratio usually recommended for spotting, to prevent matrix
precipitation upon mixing with the aqueous desalting solution
wetting the ZipTip. The higher than 50% (v/v) content of
organic solvent in the spotting solution enables spotting smaller
volumes (0.5
μ
L of
α
L or less) as organic solvents spread easily on
metal surfaces, and it also reduces drying time. Proper crystal-
lization exhibits milky amorphous spots with equally sized
rounded crystals visible under a microscope, evenly distributed
on the plate without clumping. Touching the plate surface
with a ZipTip during spotting is not recommended as it often
results in uneven crystallization and clumping. As a general
guidance, sample transfer and solvent evaporation should be
avoided during peptide extraction or peptide losses will occur.
This was demonstrated in a study by Speicher et al. (
11
) using
radiolabeled proteins by systematically evaluating peptide
recoveries after in-gel trypsin digestion. It was found that
approximately 80% of the labeled tryptic peptides could be
extracted from gel bands containing 1-10 pmol of protein,
and at least 70% could be extracted at 200- to 500-fmol levels.
It was demonstrated that although 70-85% of tryptic peptides
could be extracted from gels over a range of conditions and
protein concentrations, further extraction attempts resulted in
substantial additional losses. Even minimal handling resulted
in adsorption loss of about 10-15% of extracted peptides.
Adsorptive losses to plastic surfaces were particularly high,
sometimes greater than 50%, and were variable if extracts were
partially dried in a SpeedVac to concentrate the sample or to
remove ACN. Although the ZipTip purifi cation procedure is
labor-intensive, it provides consistently good MS data quality
and sensitivity in MALDI MS. CHCA MALDI matrix solutions
can elute peptides from C18 ZipTips directly onto a MALDI
plate, and the thorough mixing of peptides with the matrix
creates excellent matrix crystallization conditions. It eliminates
μ
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