Biology Reference
In-Depth Information
3.9. Spot Identifi cation
by Mass Spectrometry
With the picked spots, proceed according to standard procedures
for in-gel digestion, peptide elution, and peptide purifi cation.
A fi nal ZipTip purifi cation is highly recommended. Analyze spots
by ESI or MALDI tandem mass spectrometry. For data analysis,
include a modifi cation for each cysteine residue for the Cy3 fl uoro-
phore (Monoisotopic mass addition: 672.30 Da; Average mass
addition: 672.83 Da).
4. Notes
1. If the use of a different lysis buffer is inevitable, strictly avoid
the inclusion of thiols (e.g., dithiothreitol (DTT), dithioeryth-
ritol (DTE),
-MSH)) or primary amines
(e.g., from Pharmalytes or carrier ampholytes added to the
buffer), since these compounds act as competitors for the satu-
ration dye.
2. Vascularized tissue frequently contains endogenous blood to a
varying degree from sample to sample. This represents a con-
siderable bias, possibly leading to false positive proteins erro-
neously characterized as proteins of different abundance. As a
consequence, check each gel for the presence of a characteristic
array of albumin spots in the 2D gel at a molecular mass of
around 60 kDa. Presence of these spots requires a careful con-
sideration whether protein spots present at different abundance
in the samples may be the result of a different degree of vascu-
larization in individual samples. Investigate appropriate data-
bases to rule out typical serum proteins.
3. Total protein content may vary considerably between different
biological materials. As a very rough fi rst-order approximation,
assume a total extractable protein amount of 100 pg per somatic
mammalian cell from cell culture and 25
β
-mercaptoethanol (
β
g of protein/mg tis-
sue sample. Thus, to prepare a lysate containing 5
μ
μ
g protein,
50,000 cells or 0.20 mg tissue sample are required.
4. If protein spot identifi cation from a preparative gel has to be
performed, make sure lysis conditions can be scaled up to the
required protein amount in a volume compatible with your iso-
electric focusing device. Switching to different 2D PAGE
equipment for preparative gels must be avoided, since devia-
tions in the spot pattern might impair the correct spot alloca-
tion between analytical and preparative gels. In addition, beware
of protein concentrations exceeding 5
L, since lysates may
get signifi cantly depleted of poorly soluble proteins.
5. In cases were substantial amounts of sample have remained
undissolved, add fresh lysis buffer and repeat the lysis procedure
once or twice. Repeated extraction of the pellet with fresh
μ
g/
μ
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