FR AB - Science Reference

emove contaminants. The bound proteins are subsequently
digested with trypsin. The beads retain peptides
during digestion and may be deposited directly
on a target for matrix assisted laser desorption/ionization
(MALDI). On-target washing may also be performed
to remove substances inhibiting ionization.
The method provides high-quality peptide mass fingerprint
data for protein samples of only 100 nM.
PROTEINS—PURIFICATION
AND CHARACTERIZATION
Zuo X, Speicher DW. Quantitative evaluation of protein
recoveries in two-dimensional electrophoresis with
immobilized pH gradients. Electrophoresis 2000;21:3035–
3047.
Protein recoveries are systematically evaluated at
the various steps of separation by two-dimensional
gel electrophoresis using immobilized pH gradients
(IPGs) for the isoelectric focusing stage. The Multiphor
and IPGphor systems from Amersham Pharmacia
Biotech (Uppsala, Sweden) are used for the separation
of metabolically radiolabeled proteins from
Escherichia coli. With the Multiphor system, in which
sample application to the IPG strips and subsequent
electrofocusing are performed in separate vessels,
sample application from cups gives better protein
recoveries than sample loading by IPG rehydration. In
the IPGphor system, in which sample loading and
electrofocusing are performed in the same vessel, the
rehydration method gives better recoveries. In all
cases, carrier ampholytes in the sample buffer increase
protein recoveries. Thiourea does not affect
protein recoveries, but does improve protein resolution
during electrofocusing.
Zuo X, Speicher DW. A method for global analysis of
complex proteomes using sample prefractionation by
solution isoelectofocusing prior to two-dimensional
electrophoresis. Anal Biochem 2000;284:266–278.
Increasing the protein load on immobilized pH
gradient (IPG) strips in an attempt to improve the
number of visible spots resolved by two-dimensional
electrophoresis is limited by the tendency of proteins
to aggregate or precipitate when high protein loads
are used, causing smearing in the isoelectric focusing
dimension. The use of solution-phase isoelectric focusing
to minimize such smearing is discussed, and an
isoelectric focusing apparatus for prefractionating proteins
for this purpose is described.
ARTICLE WATCH
Gygi SP, Corthals GL, Zhang Y, Rochon Y, Aebersold R.
Evaluation of two-dimensional gel electrophoresis-based
proteome analysis technology. Proc Natl Acad Sci USA
2000;97:9390–9395
It is widely assumed that two-dimensional gel
electrophoresis, coupled with mass spectrometric
identification of spots, enables a broad coverage of
the proteome. Consistent with this view is the observation
that the number of spots observed by twodimensional
electrophoresis approaches the number
of proteins predicted for a given organism or tissue.
This study examines an arbitrarily selected section
from a narrow pH range (4.9–5.7) two-dimensional
display of yeast proteins in which more than 1500
spots are visualized by silver staining. Fifty spots were
digested with trypsin and analyzed by liquid chromatography
combined with tandem mass spectrometry,
and a total of 39 different proteins were identified
from a region where the yeast genome is predicted to
encode 57 gene products. However, these represent
only the proteins predicted on the basis of codon
usage to be the most abundant. Furthermore, only 14
of the proteins identified were predicted to appear in
the analyzed section. Additionally, single genes produced
multiple spots, and the products of up to six
genes were found to make up a single spot, presenting
problems for both quantitative protein expression
comparisons and database-matching studies. It is concluded
that the method is unsuited for the analysis of
low-abundance proteins and that statements about
the feasibility and straightforwardness of proteome
analysis based on the two-dimensional electrophoresis
mass spectrometry strategy should be rethought.
[This article has also received comment by Smith RD.
Probing proteomics—seeing the whole picture. Nat
Biotechnol 2000;18:1041–1042.]
Ghaemmaghami S, Fitzgerald MC, Oas TG. A quantitative,
high-throughput screen for protein stability. Proc
Natl Acad Sci USA 2000;97:8296–8301
The use of mass spectrometric measurements of
hydrogen/deuterium exchange to compare the rates of
denaturation of different but related proteins is
described. Matrix-assisted laser desorption/ionization
(MALDI) is employed to measure the rates of exchange
of amide hydrogen atoms with deuterium in
solution as a function of denaturant concentration to
provide a measure of protein stability. The method
permits large numbers of protein samples to be
screened in a short time, does not demand that the
proteins of interest are pure or available in large quantities,
and is forgiving of the presence of denaturants.
Mutants of the � repressor are studied to illustrate the
JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 11, ISSUE 4, DECEMBER 2000 183