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60
50
Retentate
Filtrate
40
30
20
10
0
Mass Bin
FIGURE 3
Mass distributions of peptides observed in the filtrate (blue) and retentate (red) fractions of peptide products of
an acid cleavage digestion.
(Modi
ed from reference #28.)
Quantitation Using Isotope Ratios
An important aspect of any robust proteomic
method is its ability to be incorporated into
a quantitative work
a twofold increase in the number of viruses in
the longer incubation (
Figure 4
). As usual, care
should be taken when using a label that provides
only a 2Da isotope increment.
33
ow. Asp-selective micro-
wave-supported acid hydrolysis can be carried
out under conditions that introduce one atom
of
18
O fromH
18
O heavy water into peptide prod-
ucts.
13
Adenovirus from Hela cells was digested
with 12.5% acetic acid for 90 seconds at 140
C.
Both formic and acetic acids were evaluated,
and digestion in formic acid was found to intro-
duce multiple atoms of
Branched Proteins: SUMOylation
Branched proteins are formed by isopeptide
bonds between the
e
NH
2
group of an exposed
Lys residue on the substrate protein and the C
terminus of modifying proteins such as ubiquitin,
SUMO, or NEDD8. Tryptic digestion of a SUMO--
modi
18
O. Tandem MS was
ed protein leaves between 19 and 32 resi-
dues (depending on which SUMO protein) of
the SUMO branch attached to a substrate peptide
that is usually shorter. The long SUMO branch
dominates fragmentation in tandem mass spec-
trometry experiments. Osula and colleagues
simpli
used to con
rm the location of isotope incorpo-
ration, on the C terminus in most of the peptide
products. Digestions of adenovirus isolated from
a standard number of HeLa cells at 24 and 48
hours after infection were carried out in light
and heavy water, respectively. The samples
were
ed the structure of the target branched
peptide by incorporating microwave-supported
combined and analyzed to con
rm
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