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"Sample Preparation Issues in Proteomic Analysis"
Thursday January
24th, 2002
Lynx Therapeutics
Hayward, CA
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here for presenter bios
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here for poster abstracts
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Schedule
4-5 pm coffee and conversation, poster viewing
5-5:30 Karin Rodland, Pacific Northwest National Laboratory
5:40-6:10 Martin Schurenberg, Bruker Daltonik GmBH
6:15-6:45 Diether Recktenwald, BD Biosciences
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Abstract |
"Partial Purification
of Membrane Proteins from Ovarian Tumor Cells for
Proteomic Analysis" by
Karin D.
Rodland (presenter), David Springer, David
Wunschel and Richard
Zangar,
Pacific Northwest National Laboratory
karin.rodland@pnl.gov
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Proteins expressed in
and on the plasma membrane of mammalian cells play a key
role sensing the cell's extracellular environment and
modulating cellular behavior in response to changes in
that environment. In
particular, acquisition of unregulated proliferation,
invasiveness, and metastatic capability by tumor cells
is likely to reflect changes in the expression of
specific plasma membrane
proteins such as growth factor receptors, adhesion
molecules and cellular proteases. We have begun an
investigation of changes in plasma membrane protein
expression in human ovarian cancer lines associated with
progression from anchorage-dependent primary tumors to
anchorage-independent invasive tumor cells growing as
ascites. However, defining the range of plasma membrane
proteins expressed by tumor cells presents several
technical challenges, including purification of plasma
membrane proteins from other cellular compartments,
solubilization of integral membrane proteins, post-translational
modifications of membrane proteins including
glycosylation and phosphorylation, and compatibility of
the plasma membrane preparation with mass
spectrometry-based analysis methods.
We have developed a procedure for partial
purification of membrane proteins based on a combination
of differential centrifugation, affinity chromatography,
and anion exchange chromatography.
This procedure is compatible with MALDI, LCQ-MS-MS
and FTICR-MS and can be used for accurate mass tag
determination. Using
this procedure we have succeeded in identifying a
variety of integral membrane proteins, including
receptor tyrosine kinases, seven-transmembrane domain
receptors, membrane-associated ligands, proteases, and
phosphatases, as well as adhesion molecules and
histocompatibility
proteins. |
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"Improved Sensitivity, Speed and Success Rate for
Protein Identification by MALDI-MS
is Achieved by Targets Containing
Hydrophilic Anchors" by Martin
Schürenberg1 (presenter),
Detlev Suckau1, Peter
Hufnagel1 and Eckhard
Nordhoff2,
1Bruker Daltonik
GmbH, 2Max-Planck-Institute
for Molecular Genetics
msch@bdal.de
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We developed new
sample targets for MALDI mass
spectrometry that simplify
high-throughput analysis of
biomolecules as peptides, proteins,
and oligonucleotides and
significantly improve the detection
limits. These MALDI targets are
coated with a hydrophobic layer.
Embedded in this hydrophobic surface
are non-coated spots with
well-defined size between 200 and
800 µm diameter, which represent
hydrophilic sample anchors. Each
transferred sample drop contacts one
anchor and this anchor causes the
sample drop to shrink exactly to the
dimension of the anchor area after
evaporation of the solvent due to
the strong water repellent nature of
the hydrophobic surface. As a result
of the sample concentration step
during solvent evaporation the
detection sensitivity is improved
approximately 10-100-fold. The small
sample area also reduces the need
for “spot hunting”
significantly, which increases the
speed of automated spectra
acquisition. Examples from model
studies as well as from real life
samples are shown, which elucidate
the capabilities of the
“AnchorChip” technique and
demonstrate the gain in information
content, sensitivity and speed of
acquisition. We will show that with
this new technology the automatic
sample preparation of few 100 µm
diameter sample areas can be done by
standard robotic systems even
without special devices like piezo-dispensers.
Optional “on-anchor” washing /
recrystallization efficiantly
removes salts / buffers in
proteomics applications. The
automatic measurement is simplified
and much faster because "spot
hunting" is no longer
necessary. Detection limits of ca.
300 amol in the fully automated
operation mode and 10 amol in the
manual mode were obtained with
AnchoChip preparations.
In addition, the observation
of an always strong increase of
information content of spectra from
the same sample suggest the
presented method to be of paramount
importance to high success rates of
high throughput protein
identification using MALDI |
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"Cell
and Organelle Purification for Proteomics" by
Bill Godfrey, Jita De, Martin Tapia, Rana Alsharif,
Sujata Iyer, Diether Recktenwald
(presenter) BD Biosciences, and Colette
Rudd, Sally
Swedberg @ThermoFinnigan
diether_recktenwald@bdis.com
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A goal of proteomics is the ability to detect all proteins in
a particular biological subsystem, such as a cell type
or cellular subfraction.
Subfractionation and purification of cellular
samples from tissues is desired for obtaining relevant
biological data about cell types or subcellular
fractions. For functional genomics methods to isolate small
numbers of specific cells followed by amplification by
the polymerase chain reaction (PCR) have been developed.
For proteome analysis large quantities of purified cells
are required, because no PCR like amplification is
available for proteins. Biological starting materials
are blood with suspended cells, which can be processed
directly for high speed fluorescence activated cell
sorting (FACS), or cell culture and tissues. Tissues
have to be dis-aggregated before FACS sorting. This is
performed mechanically and/or enzymatically. Remaining
larger aggregates and debris can be removed during the
sort by differences in light scatter properties. A
special 130um sort nozzle has been developed for FACS
sorters to optimally address the larger particles
present in disaggregated tissue and cell preparations
from cell culture. For blood cells smaller sort nozzles
are recommended. Cell subsets for sorting are identified
by their auto fluorescence, by reaction with fluorogenic
reagents like enzyme substrates or DNA reactive dyes, by
their fluorescence from fluorescent proteins from a
transfection, or by reaction with specific
fluorochromated antibodies or nucleic acid probes.
Sorted cells can be homogenized and digested for protein
analysis. For identification of cell surface proteins
intact cells were treated with proteases to digest
proteins outside of the cell membrane barrier for
protein identification by mass spectrometric analysis of
the resulting peptides. Isolated cells have been broken
into subcellular fractions like membrane, and
organelles. Subfraction particles like membrane pieces
and organelles have been sorted by FACS with the same
methods as cells to obtain highly enriched components,
which can be used for protein identification by mass
spectrometry, 2D gel electrophoresis, or on protein
arrays.
To demonstrate the technology, we have used
liquid chromatography (LC) with tandem mass spectrometry
(MS/MS) as a sensitive method for identification of
proteins from blood cell subsets highly purified by FACS.
Several million cells were sorted and subjected to
trypsin digestion for identification of cell surface
proteins by mass spectrometry. In conclusion, the
combination of magnetic and FACS cell and organelle
purification methods with LC MS/MS allows the rapid
analysis of the complete proteome of specific cells and
cellular fractions.
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