|
|
TRANSFER OF PROTEIN FROM
ELECTROPHORETIC BANDS INTO MASS SPECTROMETRY BY DIRECT ELECTROELUTION
|
|
|
|
Andreas
Chrambach, Ph.D., Principal
Investigator
Sergey Radko, Ph.D., Contractor
Zsuzsa Buzás, Ph.D., Contractor
Alfred Yergey, Ph.D., Collaborator,
LCMB
Nancy Vieira, M.S., Collaborator,
LCMB
Marcella Chiari, Ph.D., Collaborator, Institute
of Molecular Recognition Chemistry, CNR, Milan, Italy
Marina Cretich, Ph.D., Collaborator, Institute of
Molecular Recognition Chemistry, CNR, Milan, Italy
Huang-Tung Chang, Ph.D., Collaborator, National
Taiwan University, Taipei, Taiwan
|
|
|
|
The Section on Macromolecular Analysis is attempting to upgrade the common
practice of gel electrophoresis by undertaking the following projects:
the development of theory and computer programs for deriving information
from mobility regarding physical properties of analytes and polymer networks
and for predicting resolution, resolution of presently unresolved or poorly
resolved analyte types such as subcellular-sized particles and the size-
and charge-isomeric forms of peptides and their complexes, the development
of detergent and miniaturized gel methods suitable for native membrane
proteins and their complexes of a low level of occurrence, and the development
of automated analytic and preparative gel electrophoresis methods.
A significant portion of current biological research focuses on an account
of the cellular proteins responsible for the activities and functions
of the cell. One aspect of that research, designated proteomics, is working,
by means of mass spectrometry, toward the identification of proteins on
the basis of their mass. Conventional practice calls for separating the
mixture of proteins in the cellular extract of interest by two-dimensional
gel electrophoresis and detecting the proteins as "spots" by
specific staining methods. The spot of interest is then excised from the
gel, the gel slice is dehydrated and reswollen in the presence of the
proteolytic enzyme trypsin, and the tryptic peptides derived from the
protein are eluted from the gel slice for measurement of their mass by
mass spectrometry. The mass of the peptides is then compared with that
of aminoacid sequences in databases stored in computer memory and derived
from the corresponding DNA sequence. Such identification therefore depends
on a previous genetic analysis of the cellular material in question.
Our laboratory developed an alternative procedure for collecting protein
identified as a gel band (or spot); it replaces band excision with direct
electroelution of the intact protein by placing electrodes across the
band at a direction orthogonal to that of electrophoretic migration in
the separating gel (Figure 10). The alternative procedure offers several
advantages over conventional practice. First, it is applicable to those
cases in which no earlier DNA sequence analysis has been performed and
for which, therefore, no sequence exists in the database. Second, even
where a sequence has been determined, the procedure adds the mass of the
intact protein to that of its constituent peptides for purposes of identification.
Third, it allows for electrofocusing (IEF) and critical re-electrophoresis
analysis of the eluate before mass spectrometry. Fourth, it avoids the
presence of gel during tryptic digestion, thus increasing the rate of
digestion and avoiding adsorption of protein, peptides, and enzyme. Fifth,
it appears more adaptable than an excision procedure to automation of
sequential spot elution. We were able to verify the first three advantages
while the last two remain hypothetical. The primary potential importance
of such an approach, compared with conventional practice, lies in the
fact that the electroelution of the intact protein, when applied to Immobiline
IEF gel strips of high load capacity, allows for an accumulation of large
protein loads for a subsequent two-dimensional electrophoretic separation
with a number of narrow pH gradients in the first dimension and subsequent
peptide analysis of the bands. The alternative approach will allow for
a significant increase in the number of proteins detected by two-dimensional
gel electrophoresis, which currently yields only 10 to 20 percent of proteins,
i.e., those that are most abundant. Of further interest for the future
of proteomics is the fact that we have achieved a direct electroelution
of a native protein from a nondetergent gel. This finding foreshadows
the possibility of identifying those post-translationally modified proteins
that on SDS-PAGE migrate together.
Figure 10
Apparatus for a direct orthogonal electroelution
of gel electrophoretic protein bands.
Sequential electroelution of protein
bands on the two-dimensional gels remains to be solved. A possible solution
may be to polymerize on a net, which is permeable to the orthogonally
directed current of electroelution. A suitable net support has been designed
and constructed. We intend to position the gel by moving the net support
under micromanipulator control, thereby preventing gel stretching as we
move sequentially from spot to spot. Once a suitable frame for the net-supported
gel is constructed, the positioning of the net will have to be interfaced
with the image analysis system to arrive at a computer-controlled automated
protein elution method without any gel sectioning.
|
|
PUBLICATIONS
- Buzas
Z, Chang HT, Vieira NE, Yergey AL, Stastna M, Chrambach A. Direct
vertical electroelution of protein from a PhastSystem band for mass
spectrometric identification at the level of a few picomoles. Proteomics
2001;1:691-698.
- Buzas
Z, Li T, Chrambach A. Horizontal gel electrophoresis of SDS-proteins
on the PhastSystem with an at least 25-fold increased protein load volume.
Anal Biochem 2001;292:161-163.
- Chang
HT, Yergey AL, Chrambach A. Electroelution of proteins from bands
in gel electrophoresis without gel sectioning for the purpose of protein
transfer into mass spectrometry: elements of a new procedure. Electrophoresis
2001;22:394-398.
- Chiari
M, Cretich M, Stastna M, Radko SP, Chrambach A. Rapid capillary
coating by epoxy-poly-(dimethylacrylamide): performance in capillary
zone electrophoresis of protein and polystyrene carboxylate. Electrophoresis
2001;22:656-659.
- Chrambach
A, Chrambach A, Brining SK. Gel electrophoretic distinction between
Congo Red nonreactive beta-amyloid (1-42) and beta-amyloid (1-40). Electrophoresis
2000;21:760-761.
- Chrambach
A, Radko SP. Size-dependent retardation and resolution by electrophoresis
of rigid, submicron-sized particles, using buffered solutions in presence
of polymers: a review of recent work from the authors' laboratory. Electrophoresis.
2000;21:259-265.
- Gianazza
E, Sirtori CR, Castiglioni S, Eberini I, Chrambach A, Rondanini A, Vecchio
G. Interactions between carbonic anhydrase and its inhibitors revealed
by gel electrophoresis and circular dichroism. Electrophoresis 2000;21:1435-1445.
- Gombocz
E, Chrambach A, Yefimov S, Yergey AL. Electroelution of nonfluorescent
stacked proteins detected by fluorescence optics from gel electrophoretic
bands for transfer into mass spectrometry. Electrophoresis 2000;21:846-849.
- Li
Y-M, Chrambach A. Gel electrophoretic isolation in the hundred microgram
range of recombinant SDS-syntaxin from sea urchin egg cortical vesicles.
Prep Biochem Biotechnol 2001;31:369-387.
- Radko
SP, Stastna M, Chrambach A. Capillary zone electrophoresis of sub-micron-sized
particles in electrolyte solutions of various ionic strengths: size-dependent
electrophoretic migration and separation efficiency. Electrophoresis
2000;21:3583-3592.
- Radko
SP, Stastna M, Chrambach A. Relation of peakwidth to polydispersity
of liposome preparations subjected to capillary zone electrophoresis.
J Chromatogr B Biomed Sci Appl 2001;761:69-75.
- Radko
SP, Stastna M, Chrambach A. Size-dependent electrophoretic migration
and separation of liposomes by capillary zone electrophoresis in electrolyte
solutions of various ionic strengths. Anal Chem 2000;72:5955-5960.
- Stastna
M, Radko SP, Chrambach A. Discrimination between peak spreading
in capillary zone electrophoresis of proteins due to interaction with
the capillary wall and due to protein microheterogeneity. Electrophoresis
2001;22:66-70.
- Stastna M, Radko SP, Chrambach A. Separation efficiency in protein
zone electrophoresis performed in capillaries of different diameters.
Electrophoresis 2000;21:985-992.
- Yefimov
S, Sjomeling C, Yergey AL, Chrambach A. Stacking of unlabeled sodium
dodecyl sulfate-proteins within a fluorimetrically detected moving boundary,
electroelution and mass spectrometric identification. Electrophoresis
2001;22:999-1003.
- Yefimov
S, Sjomeling C, Yergey AL, Li T, Chrambach A. Recovery of sodium
dodecyl sulfate-proteins from gel electrophoretic bands in a single
electroelution step for mass spectrometric analysis. Anal Biochem 2000;284:288-295.
- Yefimov
S, Yergey AL, Chrambach A. Sequential electroelution and mass spectroscopic
identification of intact sodium dodecyl sulfate-proteins labeled with
5(6)-carboxyfluorescein-N-hydroxysuccinimide ester. Electrophoresis
2001;22:2881-2887.
- Yefimov S, Yergey AL, Chrambach A. Transfer of SDS-proteins from gel
electrophoretic zones into mass spectrometry, using electroelution of
the band into buffer without sectioning of the gel. J Biochem Biophys
Methods 2000;42:65-78.
|
|
|
|