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the dephosphorylated lysate to an aliquot of the original sample, it
is possible to identify which proteins contain these specifi c phos-
phorylated amino acids (
1
). Alternatively, with the development of
labeling chemistries, it is increasingly possible to label specifi c
PTMs directly with a reactive fl uorophore. Here, we demonstrate
oxidized protein labeling utilizing a probe that specifi cally reacts
with carbonylated amino acids, a stable indicator of protein oxida-
tion. Furthermore, PTMs may be determined by fi rst modifying
the PTM and subsequently labeling with existing chemistries. The
ubiquitination and palmitoylation labeling protocols presented
here, and the modifi ed “biotin-switch” method to label S-nitrosylation
(
2
), are examples of this strategy.
Our preferred experimental design involves rotating the NHS-Cy3
and NHS-Cy5 labels within each experimental group (Fig.
1a
);
such that samples from each group are labeled with both fl uoro-
phores (
3
). In contrast, the fl uorophore used to label the internal
control (IC) remains constant (NHS-Cy2). The IC is a pooled
sample containing an identical amount of protein of each sample in
the study (
3, 4
).
However, in some cases, it is not possible to rotate the fl uoro-
phores. For example, the method for studying oxidized proteins
described below uses an AlexaFluor 488 (AF-488) conjugated
label that induces a shift in pI compared to that seen with NHS-Cy3
and NHS-Cy5. In addition, the AlexaFluor 647 label that is also
available induces a different degree of shift. Therefore, a single-
wavelength label for oxidation (AF-488) is used with all samples in
1.1. Experimental
Design
Fig. 1. Experimental design. (
a
) With our preferred design, the internal control (IC) is con-
sistently labeled with NHS-Cy2. In contrast, NHS-Cy3 and NHS-Cy5 alternate between the
experimental groups. (
b
) When labeling oxidized and total proteins, rotating the labels is
not possible. The oxidized samples must remain labeled with AlexaFluor 488 (AF-488), the
IC with NHS-Cy5, and the total protein samples with NHS-Cy3.
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