FR AB - Science Reference

P69-T
Efficient detergent and Coomassie blue dye removal from peptide
samples prior to MALDI-TOF MS.
W. Kopaciewicz, E. Kellard; Millipore, 17 Cherry Hill Drive, Danvers,
MA 01923
Advancements in Mass Spectrometry (MS) instruments over the last 5 years
have reduced the amount of sample required and expanded the molecular
weight analysis range. As such, MS is an increasingly valuable tool for the
analysis of biomolecules in areas of proteomics and functional genomics.
However, the presence of salts, detergents and dyes can decrease macromolecular
ionization and/or obscure mass peaks, thus decreasing instrument
performance. As such, numerous methods and devices have been
developed for sample preparation prior to MS analysis.
Millipore ZipTip sample preparation devices containing C18 and C4 media
reversed phase media have been demonstrated to be effective for the concentration
and desalting of microliter volumes of peptide, protein and
oligonucleotide samples prior to MALDI-TOF MS. Although reversed phase
chromatography meets the vast majority of the sample preparation needs,
there are cases where it isn’t totally effective. This is especially evident when
the contaminants have similar hydrophobic character as the solutes and thus
co-purify.
Alternatively, a subset of normal phase chromatography termed hydrophilic
interaction chromatography has been shown to be an efficient sample preparation
technique for certain biomolecules in those cases where reversed
phase struggles (1,2,3). The method utilizes a highly hydrophilic adsorbent
that binds sample out of a high organic mobile phase (e.g. 90% acetonitrile).
After washing with this solvent, bound molecules are then eluted by decreasing
the organic content (e.g. 50% acetonitrile). In this poster, we demonstrate
the use of a ZipTip with hydrophilic interaction media (ZipTipHPL). The
device was very effective for the removal of detergents and coomassie blue
dye from sub-picomole quantities of peptides. The resulting mass spectra
demonstrated good signal strength and fidelity of peptide capture.
1. Alpert, A.J., J. Chromatogr. 499 (1990) p.177
2. Zhu, B.Y., et al., J. Chromatogr. 548 (1991) p.13
3. Scherer, J.P., et al., Anal. Biochem. 215 (1993) p.292
P71-M
Factors affecting rapid sample processing for protein capture
chips analyzed by MS.
J. Chan1, P.T. Jedrzejewski1, P. Wagner1, S. Nock2; 1Zyomyx, Inc,
3911 Trust Way, Hayward, CA 94545, 2Zyomyx, Inc.
Robust, sensitive, and comprehensive methodologies for systemic approaches
to proteome analysis analogous to genomics methods (e.g., gene chips) are
not available. Current methodologies (e.g., 2D-PAGE and multi-dimensional
chromatographic methods) suffer from fundamental limitations. In order to
overcome these limitations, we as well as others have focused on the development
of alternative methods (e.g., protein capture chips) for proteomics.
Protein capture chips, consisting of arrayed capture molecules (e.g. protein,
DNA, or small molecule), allow for rapid and comprehensive micropurification
and analysis of proteins. Protein chips may be readily interrogated by
mass spectrometric analysis. Data on the post-translational modifications
(PTM), protein identification, and quantitation may be obtained with a single
detection scheme without labeling of analytes.
We have been successful in microfabricating protein capture arrays, the
accompanying microfluidics devices, and implementing MS analysis. We
have investigated various parameters which have an effect on the utility and
efficacy of the overall system for proteomics. In this presentation, we will
demonstrate data on parameters affected by the type of capture molecules,
digest efficiency (e.g., time), and sensitivity of analysis (e.g., LC-MS conditions).
POSTER ABSTRACTS
ABRF 2001 ABSTRACTS
P70-S
Improved proteome coverage through the use of affinity tags
with nanoscale capillary LC/MS/MS.
K. Blackburn1, W. Burkhart2, R. Davis2, M. Moyer2, A. Moseley2; 1Glaxo Wellcome, 5 Moore Drive, PO Box 13398, Research Triangle Park,
NC 27709, 2Glaxo Wellcome
Because of the inherent difficulties associated with 2D gel electrophoresis
(low throughput, bias against certain classes of proteins, poor peptide recovery
from in-gel digests, etc.), numerous groups have explored ways to analyze
complex protein mixtures directly by mass spectrometry, avoiding the
gel separation altogether. In proteomic experiments where qualitative and/or
quantitative data is required for complex biological samples such as protein
complexes, organelles, or tissue samples, the number of proteins potentially
encountered may range from tens to thousands. The number of proteolytic
peptides generated from digests of such complex protein mixtures precludes
“complete” proteome coverage by any 1D-LC/MS/MS analysis. Following on
the work of Aebersold and Patterson, we have investigated the use of cysteine-modifying
“affinity” tags with avidin affinity chromatography for the
simplification of complex peptide mixtures prior to nanoscale capillary
LC/MS/MS analysis. This approach reduces the number of peptides presented
to the mass spectrometer to a more reasonable number, thus allowing
improved proteome coverage. Data will be presented on improved sample
handling procedures for labeling and digestion steps as well as qualitative
and quantitative applications to complex biological samples.
P72-T
High throughput in-gel peptide digestion and microscale sample
preparation for MALDI-MS analysis of the resulting peptide
mass fingerprint.
M.G. Pluskal1, A.M. Pitt2; 1Proteome Systems, Inc., 14 Gill St.,
Woburn, MA 01801, 2Millipore Corp.
In-gel peptide digestion has become a widely used technique for characterizing
proteins resolved by two dimensional gel electrophoresis. Peptides
generated from gel pieces are frequently contaminated with detergent and
salts. Prior to MALDI-MS analysis, these contaminants are removed using
microscale C18 sample preparation columns. In this poster, data will be presented
to demonstrate the application of a solvent resistant Multiscreen 96
well plate with an optimized low peptide binding membrane and ZipTip C18
micropipet based sample preparation. Recoveries of peptides (Mz range of
1000 to 5000 Dalton) derived from standard protein protease digests, were
estimated at various stages of the analytical process. An optimized protocol
has been established and all the reagents and consumables have been packaged
in a ready to use commercial kit. Data will be presented to show application
of this technology package to accelerate the throughput of protein
characterization by protease fragmentation.
JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 11, ISSUE 4, DECEMBER 2000 205