FR AB - Science Reference

ABRF 2001 ABSTRACTS
P57-T
Optimization of a procedure for labeling proteins with isotopecoded
affinity tags.
K. Parker, S. Pillai, S. Daniels, W. Stanick, R. Lotti, B. Purkayastha,
T. Nadler; Applied Biosystems, 500 Old Connecticut Path, Framingham,
MA 01701
The use of the isotope coded affinity tag (ICAT), developed by the laboratory
of R. Aebersold (Nat. Biotechnol. 1999 Oct 17(10):994–9), allows one
to compare the relative protein abundance between two samples by mass
spectrometry. The reagent used is essentially cysteine reactive biotinylation
reagent into which stable isotopes have been incorporated. One of the two
samples is treated with the light reagent while the other sample is treated
with the same reagent containing 8 deuterium atoms such that the samples
are chemically equivalent but differ only in mass. In this way two peptides
may be monitored in the mass spectrometer and quantified relative to one
another by peak intensity.
A number of imporant questions concerning the use of this technique are
addressed in this presentation. The reagent has been extensively characterized
and the recommended protocol for its use has been optimized for ease
of use and robustness. Experiments were carried out to determine the purity,
stability, and reaction kinetics of the reagent. Additional experiments were
performed to optimize the derivatization protocol, including measurements
of the degree of alkylation obtained, determining the optimal reduction
reagent, how to remove excess reagent, and measurements of stability,
capacity, carryover, and recovery of biotinylated peptides from monomeric
avidin columns, Finally, the reproducibility of quantitative measurements in
the mass spectrometer was addressed and will be presented.
P59-M
Quantitative proteomic and mRNA expression analysis of
S. cerevisiae via metabolic labeling and multidimensional
protein identification technology.
M.P. Washburn1, G. Oshiro2, D. Wolters1, D. Schieltz1, C. Deciu1, E. Winzeler2, J.R. Yates, III1; 1Novartis Agr. Discovery Inst., 3115 Merryfield
Row Suite 100, San Diego, CA 92121, 2Genomics Inst. of Novartis Fndn.,
San Diego
Proteomic technologies are being developed to determine large-scale
changes in protein expression levels. Several quantitative proteomic methods
have recently been published where the protein expression levels are compared
between two different growth conditions by metabolic isotopic labeling
of proteins or post-growth isotopic labeling of proteins in a sample. To
fully understand how growth conditions effect biological systems, both protein
and mRNA expression levels must be analyzed. We have applied metabolic
labeling strategies to S. cerevisiae in order to determine the changes in
protein and mRNA expression levels. S. cerevisiae was grown in 15N enriched
minimal media and compared to S. cerevisiae grown in rich media. Each sample
was analyzed via mRNA expression array and multidimensional protein
identification technology. By combining these methods we were able to correlate
the changes in protein and mRNA expression levels of several hundred
gene products.
POSTER ABSTRACTS
202 JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 11, ISSUE 4, DECEMBER 2000
P58-S
Rapid genotyping using spectrometrically monitored selections.
J. Stoerker1, J.D. Mayo2, C.N. Tetzlaff3, D.A. Sarracino4, C. Richert3; 1Bruker Daltonics, 15 Fortune Drive, Billerica, MA 01821, 2Tufts Univ.,
3Univ. of Konstanz, 4Variagenics, Inc.
Presented here is an inexpensive and rapid method for genotyping single
nucleotide polymorphisms (SNPs) on PCR products using spectrometrically
monitored selection from probe libraries. We have previously reported nuclease
selections of modified oligonucleotides with increased RNA or DNA targets.
This methodology relies on the selection of short hybridization probes
complementary to the target sequence with a single-strand specific nuclease
that does not attack duplexes. Conditions are described where the matched
duplex is profoundly better protected from enzymatic digestion than single
base mismatches. Semi-quantitative MALDI-TOF MS is used to identify the
selected probes after a 10-minute digestion reaction with snake venom phosphodiesterase.
We have examined sequences from exon 10 of the cystic
fibrosis transmembrane regulatory (CFTR) gene, using a probe library representing
a number of alleles, including: native, I506S, 1651 benign polymorphism,
I506S/1651 dual polymorphism, and dF508, and dI507. Probe
libraries composed of 8-mers and 9-mers annealing to PCR products have
been used in our experiments. DNA from blood or cheekbrush samples was
used from 10 genomic sources. The method discriminates both alleles of a
heterozygote pair in a single experiment, and can discriminate alleles from
multiple loci in the same sample.
P60-T
A software algorithm for automated quantitation of ICAT labeled
peptides analyzed by mass spectrometry is described.
J. Eng, J. Ranish, R. Aebersold; Inst. for Systems Biol., 4225 Roosevelt
Way NE, #200, Seattle, WA 98105
We describe a software program which automatically generates quantitation
ratios of Isotope Coded Affinity Tag (ICAT) labeled peptides analyzed in an
LC-MS/MS experiment performed on an LCQ ion trap mass spectrometer. The
peptides are initially identified via a sequence database search. Quantitation
of each ICAT-labeled peptide pair is then performed by integrating over the
peptide’s light and heavy elution profiles. An interactive, graphical program
displays the light and heavy elution traces and allows the researcher to
adjust the area of integration as needed. This program should enable and
facilitate large scale, quantitative proteomics studies.