FR AB - Science Reference

ABRF 2001 ABSTRACTS
P49-S
4-(4-Hydroxystyryl)pyridine, a rationally designed negative ion
matrix for MALDI-TOF mass spectrometry.
J.C. Lopez, P.A. Liddell, J.W. Lippert, D.C. Brune; Arizona State Univ.,
PO Box 871604, Tempe, AZ 85287-1604
To date, most matrices for MALDI-TOF mass spectrometry have been discovered
by trial and error experiments, and through broad investigation of
compounds chemically related to known matrix molecules. The most widely
used matrices have been used to analyze proteins and other analytes in the
positive ion mode. We chose to work with negative ion matrices because this
area has received less attention, and because we occasionally analyze samples
for which a good negative ion matrix is required. In this investigation,
we synthesized and studied 4-(4-hydroxystyryl)pyridine. This compound
resembles those derived from 4-hydroxycinnamic acid in having an electronwithdrawing
moiety (in this case a pyridyl group) separated from a 4-hydroxyphenyl
moiety by a vinyl group. In the electronically excited state, charge
density delocalized across the vinyl group onto the pyridyl group will make
the latter more strongly basic, favoring abstraction of protons from both the
analyte and other matrix molecules. Preliminary experiments on Fmoc and
other modified amino acids have shown that this matrix works as well as
3-aminoquinoline, one of the best negative ion matrices reported so far. Further
comparisons involving structurally related matrix molecules are in progress
and will be reported. A characteristic for negative ion matrices is a shift
to longer wavelengths under acidic conditions.
P51-T
Advances in quantitation and identification of proteins using
isotope-coded affinity tags and LC/MS/MS using informationdependent
acquisition.
T.A. Settineri, C.L. Hunter, L. Nuwaysir, A. Patel, A. Tomaney, B. Halpern;
Applied Biosystems, 850 Lincoln Ctre. Dr., Foster City, CA 94404
Recently, Aebersold et al. introduced a method in which proteins are labeled
with an isotope-coded affinity tag (ICAT), digested, and the resulting labeled
peptides separated and analyzed by mass spectrometry. The advantage to
this technique is that the ICAT approach precludes the use of 2D gels while
allowing complete automation of the entire process. In addition, the ICAT
technique is generally applicable and can be applied to the global analysis
of protein expression. In this study we investigate the ICAT approach using
an API-QSTAR (hybrid quadrupole time-of-flight) mass spectrometer fitted
with a nano-electrospray source, ICAT reagents synthesized in-house, and
novel data reduction algorithms to both quantitate protein expression and
identify proteins from MS/MS data acquired by using information dependent
acquisition (IDA)
This approach will be demonstrated for the analysis of proteins arising from
various cell lines including E. coli and Arabidopsis treated in different ways.
After treatment with ICAT reagent and purification, samples are analyzed by
LC/MS/MS using a QSTAR mass spectrometer using information dependent
acquisition (IDA). Two new algorithms developed to identify and quantitate
ICAT expression pairs are applied to the MS scans while a third new algorithm
is applied to the MS/MS data to identify proteins. The first two algorithms
cluster the data based on ICAT fragments, and optimally collapse adjacent
spectra for maximum signal to noise. The third algorithm then identifies
the protein using the MS/MS fragment ion data to perform a database search.
POSTER ABSTRACTS
200 JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 11, ISSUE 4, DECEMBER 2000
P50-M
LC-MS of oligonucleotides using polystyrene supports and
API-TOF mass spectrometry.
D.H. Hawke1, J.S. Nelson2, R. Vinayak2, W. Xiao3, P.J. Oefner3; 1Applied
Biosystems, 850 Lincoln Ctr. Dr., Foster City, CA 94404, 2Applied
Biosystems, 3Stanford Genome Res. Ctr.
Oligonucleotides may be analyzed by either Maldi or electrospray methods
after suitable desalting of samples. Maldi methods are convenient and offer
high-speed analysis, while electrospray methods can be directly coupled to
HPLC separations. A classical application for these methods is oligonucleotide
synthesis, in this case there is usually enough material available that
sensitivity is not a major problem. LCMS (1) is advantagous if UV detection
is required, and desalting is effectively performed on-line. A newer area is
that of genotyping. In this application separation may be required prior to
analysis and current ion-pairing methods have remarkable separating power
for these molecules (2). We have coupled this separation technology to a
high-resolution API-TOF MS system (Mariner) to give both high resolution
separations and high mass accuracy measurements of oligonucleotides and
labeled oligos.
1. Huber C.G., Krajete A., Anal. Chem. (1999), 71:3730–3739.
2. Underhill, P.A., L. Jin, A.A. Lin, S.Q. Mehdi, T. Jenkins, D. Vollrath, R.W.
Davis, L.L. Cavalli-Sforza and P.J. Oefner. Genome Res. (1997), 7:996–1005.
P52-S
High-throughput SNP genotyping by MALDI-TOF MS.
M. Kostrzewa1, T. Fröhlich1, T. Wenzel1, C. Franke1, W. Pusch2, K-O. Kräuter2, Y. Stalgies2; 1Bruker Saxonia Analytik GmbH, Permoserstrasse
15, Leipzig, Saxonia D-04318 Germany, 2Bruker Daltonik GmbH,
Bremen
One of the great challenges of the upcoming postgenomic era is the determination
of sequence variations, in particular single nucleotide polymorphisms
(SNPs). These polymorphisms are believed to have an enormous
impact in diagnosis of diseases, improvement of drugs, and forensic analysis
in future. In contrast to the increasing demand for SNP genotyping, there
is still a lack of highly reliable high-throughput SNP typing techniques.
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
(MALDI-TOF MS) is a powerful advanced technology capable of the accurate
and cost-effective typing of many thousands of SNPs per day. The “GOOD
assay”, a novel sample preparation procedure for MALDI-TOF typing of
SNPs was recently presented (Sauer et al., Nucleic Acids Research, methods
online, 2000, 28, e13). This preparation uses sensitivity enhancing, chemical
modifications of the allele specific products. Main advantage of the GOOD
assay over other SNP genotyping preparation methods with MALDI detection
is that this protocol does not require solid phase purification of the products
prior MALDI analysis. SNP typing starts with a PCR encompassing a region
containing a known SNP. Thereafter dNTPs are digested with shrimp alkaline
phosphatase. In the following primer extension reaction using a charge tag
containing extension primer, a conditioned set of ddNTPs and a DNA polymerase
of allele specific products are generated. The 5� part of the extension
primer is removed by an exonuclease. By alkylation the backbone phosphate
groups are neutralised. This results in small singly charged product molecules
which are transferred onto non-protonating matrix on the MALDI target.
Mass spectra of 384 samples were acquired in less than one hour. Allele calling
was performed automatically by the novel genotools SNP manager software,
online during spectra acquisition or offline. The resulting genotypes are
stored in a table in the ASCII format together with a quality asse.