Biology Reference
In-Depth Information
utilized to label the proteins under analysis, do not affect the mass
spectrometric identifi cation process of labeled proteins. This was
demonstrated by identifying a group of Cy-dye-labeled and unla-
beled proteins.
Although MS-based protein identifi cation from 2D DIGE
gels is a well-established technique (
2
), it has multiple procedural
variations used in different laboratories. However, most of these
technical variations share the following common steps: spot pick-
ing from polyacrylamide gels, protein destaining, gel dehydration,
enzymatic digestion of the proteins and extraction of resulting
peptides, mass spectral analysis, and MS data analysis and verifi cation
of the results. The following paragraphs present a brief overview of
these steps, and more in-depth discussions are included in the
protocols section and in the reference notes.
Spot picking from polyacrylamide gels is performed from 2D gels
either manually, using a variety of tools such as pipette tips cut to
the size of the protein spot, plastic straws, metal punches, or auto-
matically using a software-controlled robotic spot picker.
1.1. Spot Picking from
Polyacrylamide Gels
1.2. Destaining
Procedures
Destaining procedures vary depending on the type of dye used to
visualize and quantitate proteins in the gel. The choice of appropri-
ate destaining procedures is important for successful MS protein
analysis. In general, gel stains can be grouped into two major cat-
egories as MS-friendly and nonfriendly. For example, a traditional
silver stain technique is sensitive, but it often requires a cumbersome
destaining procedure that may result in poor protein digestion
and poor extraction effi ciency, and hence poor MS identifi cation.
On the opposite side of the staining technique spectrum is a classic
Coomassie blue stain. It is less sensitive than silver stain but can be
readily removed from a gel spot prior to the protein digestion and
MS analysis. Colloidal Coomassie is a more sensitive version of the
Coomassie blue dye. There are also several ultrasensitive fl uores-
cent protein dyes compatible with downstream MS applications.
For example, Sypro Ruby and Krypton dyes allow visualization of
nanogram amounts of proteins and can be removed for subsequent
analysis. A major disadvantage in the use of fl uorescent dyes is in
our inability to visualize these dyes with the naked eye, hence requir-
ing fl uorescence detectors, which may be extremely expensive.
1.3. Gel Dehydration
Following the destaining step, a protein-containing gel piece is
usually dehydrated with acetonitrile and then rehydrated with a
protease enzyme solution.
1.4. Enzymatic
Digestion
and Extraction
of the Peptides
The most commonly used protease is trypsin because it produces
predictably cleaved peptides, which are within the optimal mass
range of most mass spectrometers and are, on average, long enough
to produce informative tandem MS (MS/MS) spectra for peptide
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