FR AB - Science Reference

ABRF 2001 ABSTRACTS
P33-T
A targeted approach for more sensitive and accurate protein
identification.
D.K. Leung, M.J. Horn; BioMolecular Technol., Inc., 525F Del Rey Ave.,
Sunnyvale, CA 94086
Selective isolation of peptides containing low abundance amino acids such
as tryptophan, offers a direct way to simplify protein digest mixtures prior to
mass spectrometric analysis. This approach provides benefits for both in-gel
digests and digests of protein mixtures.
To this end a solid phase reagent capable of selectively binding to tryptophan
has been developed. This solid support material binds covalently to tryptophan
or tryptophan containing peptides, allowing for their selective separation
from mixtures of peptides and proteins. Bound peptides can be released
from the solid-support by a reducing reagent to give analytical samples for
further analysis (LC, MS, etc.). The specificity of the binding chemistry coupled
with mass spectrometric analysis by MALDI affords improved accuracy
in data base search and in protein identification.
References
1. Shechter, Y., Rubinstein, M. and Patchornik, A. (1976), Biochemical and
Biophysical Research Comm. {4} (70), 1257.
2. Early, S. L., Magil, S. G., Novak, C. and Horn, M. J. (1989), Techniques in
Protein Chemistry, Academic Press, Inc., 439.
3. McEldoon, W., and Horn, M.J. (2000), 14th Protein Society Symposium,
{August}.
P35-M
Guanylation of lysines as a means to enhance confidence of protein
identification in proteomics.
S. Krishnan1, M. Lin2, K. Waddell1; 1Applied Biosystems, Foster City, CA,
500 Old Connecticut Path, Framingham, MA 01701, 2Applied Biosystems,
Framingham, MA
The completion of several genomes, including that of the human, has shifted
the emphasis to determining the proteome counterpart of biological systems.
The current approach for such a proteomic analysis is the mass fingerprinting
of tryptic digests of proteins (generated by ingel digest of 1 or 2-D gel
spots or of chromatographic fractions) using MALDI-Tof mass spectrometry
followed by database searching of the masses so obtained. It has recently
been noticed that the arginine containing peptides ionize better under MALDI
conditions compared to lysine containing peptides. We present here an
approach to use this as an advantage in enhancing confidence of the database
search matches by modifying the lysines to homoarginines. The increase
in confidence is achieved due to increase in the number of peptides seen in
the mass spectrum resulting from the guanylation of lysine e-amino group
(addition of 42 Da) and also the fact that such a modification indicates the
lysine containing peptides in the tryptic digest mix. Tryptic digests of BSA,
Enolase, alpha lactalbumin, and cytochrome C were generated by routine
procedures and mixed in various combinations. The digest mixtures were
than analyzed by MALDI-Tof mass spectrometry followed by database searching
of the peptide masses for identification of proteins. A portion of the digest
mixture was then reacted with O-methyl isourea sulfate to convert the lysines
to homoarginines. The modified digest mixtures were analyzed by MALDI-
Tof mass spectrometry followed by database searching. The analysis before
and after the modification helped identify the lysine containing peptides in
the digest and hence a more comprehensive identification of the protein mixture.
POSTER ABSTRACTS
196 JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 11, ISSUE 4, DECEMBER 2000
P34-S
Approaches to validating SEQUEST database search results.
R.E. Moore, M.K. Young, T.D. Lee; Beckman Res. Inst., City of Hope,
1450 E. Duarte Rd., Duarte, CA 91010
Tandem mass spectrometry and database searching is a very powerful
approach to protein identification. Unfortunately, the wealth of data that
makes the technique so powerful also requires extensive analyst time for data
reduction and validation of search results. When analyzing complex samples,
it can easily take more time to validate and reduce the data than it did to generate
it. This time requirement is a serious problem because the technique
is otherwise well suited to high throughput analysis.
Two aspects of result validation are analyzed: validation of search results for
individual tandem mass spectra and validation of composite results for sets
of individual spectral matches. Statistical analysis and experiments with falsified
databases demonstrate that there is a significant chance of generating
multiple incorrect matches to the same protein when examining large data
sets. This makes it imperative to validate the individual spectral matches, as
simple reliance on multiplicity of matches to a single protein does not guarantee
a correct result.
Several approaches to validating individual spectral matches were tried and
compared to the existing standard of manually comparing actual and predicted
spectra. The most successful approach was to search each spectrum
twice, once using tryptic specificity and once using no enzyme specificity,
and correlate the results. When both searches generate the same result, that
result is almost always validated by manual examination. When the non-specific
search generated a higher cross-correlation score the result of the search
using tryptic cleavage was almost never manually validated. Surprisingly,
there were a large number of searches in which the search using tryptic
cleavage resulted in a higher cross-correlation score than the search using
non-specific cleavage. This third group yielded a moderate number of spectra
that were manually validated.
P36-T
Automated analysis of single nucleotide polymorphism using mass
spectrometry.
M.S. Minkoff1, P. Ross1, L. Hall1, R. Jones2, D. Ledman1, L. Haff1; 1Applied
Biosystems, 500 Old Connecticut Path, Framingham, MA 01701, 2Inst. for
Child Hlth.
Single nucleotide polymorphisms (SNPs) are increasingly employed as
genetic markers for associated studies of many inherited diseases and traits.
It is apparent that approximately 3 million putative SNPs will be found in the
Human Genome. In order to tackle the task of validating the significance of
any of these putative SNPs the chemistry and analytical platforms must meet
demanding throughput and cost requirements. The SNP assay developed to
meet these criteria incorporates the use of simple, inexpensive primers and
unlabeled nucleotides for single base extensions, thus keeping down the cost
of analysis. Multiplexing the analysis of SNPs enhances affordability of genotyping
studies as well as significantly improving throughput. The use of
high throughput automated Mass Spectrometry with integrated data acquisition,
genotyping, sample handling software package simplifies and speedsup
the task. In addition the top-level software application package tracks
sample information throughout the workflow and stores all information in a
central database. Data in support of multiplexed, automated sample handling,
acquisition, analysis and reporting is presented.