“Out-Gel” Tryptic
Digestion Procedure
for Chemical Cross-Linking Studies with Mass Spectrometric Detection
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Abstract
SDS-PAGE is one of the most powerful
protein separation techniques,
and in-gel digestion is the leading method for converting proteins
separated by SDS-PAGE into peptides suitable for mass spectrometry-based
proteomic studies. In in-gel digestion, proteins are digested within
the gel matrix, and the resulting peptides are extracted into an appropriate
buffer. Transfer of the digested peptides to the liquid phase for
subsequent mass spectrometric analysis, however, may be hampered by
peptide-specific characteristics, including size, shape, poor solubility,
adsorption to the polyacrylamide, andin the case of cross-linking
applicationsby the branched structure of the peptides produced.
This can be a limitation in cross-linking studies where efficient
recoveries of the cross-linked peptides are critical. To overcome
this limitation, we have developed a modification to the standard
in-gel digestion procedure for SDS-PAGE-separated cross-linked proteins,
based on older passive diffusion methods. By omitting the gel staining
and gel fixation steps, intact proteins or cross-linked protein complexes
can move through the gel and into the buffer solution. Digestion of
the entire protein in the buffer <i>outside</i> the gel
increases the probability that most of the proteolytic peptides produced
will be present in the final digest solution. The resulting peptide
mixture is then freed of SDS and concentrated using SCX (strong cation
exchange) zip-tips and analyzed by mass spectrometry. For standard
protein identification studies and the recovery of noncross-linked
peptides, the in-gel procedure outperformed the out-gel procedure,
but for cross-linking studies with enrichable cross-linkers (such
as CBDPS), the standard out-gel procedure allowed the recoveries of
cross-links not recovered via the in-gel method. Labeling experiments
showed that, with an enrichable cross-linker, 93% of the cross-links
showed better or equal recoveries with the out-gel procedure, as compared
to the in-gel procedure. It should be noted that this method is not
designed to <i>replace</i> in-gel digestion for most proteomics
applications. However, by using the out-gel method, we were able to
detect twice as many interprotein CBDPS cross-links from the histone
H2A/H2B complex as were found in the in-gel digested sample