FR AB - Science Reference

P61-S
Genopure: a novel magnetic bead DNA purification system for
MALDI-TOF MS analysis.
M. Kostrzewa1, J. Bimmler2, I. Thomas2, T. Wenzel1, E. Nordhoff3, H. Rauth3, T. Fröhlich1; 1Bruker Saxonia Analytik GmbH, Permoserstrasse
15, Leipzig, Saxonia D-04318 Germany, 2Bruker Saxonia Analytik
GmbH, Leipzig, 3Max Planck Inst. of Molec. Genet., Berlin
MALDI-TOF mass spectrometry has a high potential for high throughput DNA
analyses because of its accuracy, speed, automation capabilities, and costeffectiveness.
Unfortunately, molecular biological reactions are performed in
the presence of high amounts of salts and detergents which form adducts
with DNA or interfere with matrix crystallization, respectively. Therefore,
effective DNA purification is indispensable for successful and valid MALDI-
TOF MS analysis. We present a novel magnetic DNA purification system
which gives excellent results in subsequent mass spectrometric measurement.
Small double stranded PCR products were precipitated on paramagnetic
beads using an appropriate binding solution. The binding of DNA to the particles
did not require any labeling of primers or dNTPs. After several washing
steps with buffers containing either ethanol or isopropanol, DNA was
eluted and measured with 3-HPA matrix in a MALDI-TOF mass spectrometer.
Residual PCR primers were removed while PCR products down to about
50 bp in size were recovered with high yield. Quality of the acquired spectra
was superior to that of alternative purification protocols.
Alternatively, eluted PCR-Products were used for subsequent reactions like
primer extension or restriction enzyme digest. Primer extension products or
very small restriction enzyme digest products (about 20 bp) were again purified
by the magnetic bead system to MALDI quality with high recovery using
a dedicated binding buffer. The combination of both purification procedures
enabled the genotyping of SNPs by performing PCR, purification of the
PCR product, primer extension or digest, and clean-up of the allele specific
products in one reaction tube. High quality spectra could be acquired for several
model systems allowing the unambiguous determination of genotypes.
The bead handling can easily be automated on standard pipetting robots
thereby enabling the processing of thousands of samples per day.
P63-T
Bioinformatics assessment of mass spectrometric chemical
derivatisation techniques for proteome database searching.
K.S. Sidhu1, P.J. Lester1, S.J. Gaskell1, O.V. Wolkenhauer1, S.G. Oliver2, F. Brancia1, A.G. Sullivan1, P. Sangvanich1, S.J. Hubbard1; 1UMIST, UK,
PO Box 88, Manchester, Lancashire M60 1QD, United Kingdom,
2Manchester Univ.
Identification of proteins from the mass spectra of peptide fragments generated
by proteolytic fission and subsequent database searching is one of the
most powerful techniques in proteome science. Using computer simulation,
we have studied how various chemical derivatisation techniques developed
in the Michael Barber Centre for Mass Spectrometry at UMIST can improve
the efficiency of protein identification from mass spectrometric data. The
impact of these different derivatization strategies, which promote and stabilise
certain fragmentation pathways yielding additional database search
information has been assessed. For example, by reliably promoting fragmentation
at aspartic acid residues after homoarginine derivatisation, 85% of
yeast proteins can be unambiguously identified from a single peptide with a
measured mass accuracy of 500 ppm by using the associated fragment ion
data. We present results here for 3 alternative techniques, both for single proteins
and for simple mixtures. Additional data have also been generated to
compare these “partial sequencing” methods with high accuracy mass spectrometry
where peptide mass accuracy can be achieved at 50 ppm and
beyond. Interestingly, for protein mixture analysis, the inclusion of limited
sequence information for the peptides can compensate and exceed the
search efficiency available via high accuracy searches of around 20 ppm, suggesting
the particular use of this experimental approach for the simple protein
mixtures that are routinely obtained from 2D-gels.
POSTER ABSTRACTS
ABRF 2001 ABSTRACTS
P62-M
Industrial scale high throughput proteomics.
J. Brown, D. Gostick, P. Young, J. Langridge; Micromass UK Ltd,
Floats Road, Wythenshawe, Manchester M23 9LZ, United Kingdom
With the completion of the Human Genome sequence MALDI-TOF-MS is
increasingly becoming an established method for identification of proteins
separated by 2D gel electrophoresis. Mono-isotopic peptide mass fingerprinting
(PMF) has been previously shown to be amenable to full automation
encompassing the process of acquisition, data processing and databank
searching under full software control.
Until now the throughput of MALDI-TOF-MS for proteomics has been limited
to several hundred samples in a working day and this represents approximately
5–10% of the total proteins resolved by a large format 2D gel. To
reduce the number of proteins to be identified the 2D gels are imaged and
analysed to determine differences in expression levels within a set of gels.
Although much of the image processing is semi-automated the comparison
is labour intensive as manual pattern matching has a role in the gel alignments
(land marking). Increased MS sample throughput allows the possibility
of identifying every protein spot in a 2D gel within a day. This could eliminate
the potentially erroneous step of human gel image alignment, whereby
land marking could be achieved using the MS data.
Increased sample throughput requires greater capacity and robust unattended
instrument operation. In this poster we describe an integrated robotic
multiple plate loader that allows overnight unattended MS operation. Other
improvements include an increased laser repetition rate that allows the data
capture rate to increase four fold. Sample tracking, data archiving and data
reporting are essential attributes of this new technology and these aspects are
outlined in the present
P64-S
Automated high throughput protein identification on a hybrid
quadrupole orthogonal acceleration time-of-flight mass
spectrometer coupled with a MALDI ion source.
A. Millar, R. Tyldesley, J.I. Langridge, J.B. Hoyes, Y. Philip, R. O’Malley,
R. Bateman; Micromass UK Ltd., Floats Road, Wythenshawe, Manchester
M23 9LZ, United Kingdom
The mass spectrometry technique providing the highest throughput in terms
of samples per hour, is currently MALDI-TOF-MS. This technique provides a
peptide mass fingerprint of the protein digests and allows rapid and accurate
identification of the parent protein by comparison to a databank. However,
under some circumstances, for example if the number of peptides detected
is small or if the sequence coverage is poor, it is advantageous to be able to
include even a short piece of sequence information to provide added specificity.
In a conventional MALDI-TOF-MS instrument post source decay (PSD)
can be used to try and generate sequence information, however this
approach is notoriously unreliable in producing good quality MS/MS data.
One reason for this is that the peptide does not undergo fragmentation in a
controlled environment such as a gas cell.
An alternative approach is to couple a MALDI ion source to a hybrid quadrupole
orthogonal acceleration time-of-flight (Q-Tof) mass spectrometer, which
results in predictable fragmentation. In contrast to a conventional MALDI-
TOF-MS instrument the resolution and mass measurement accuracy of the
data is comparable between the MS and MS/MS modes. This allows superior
data acquisition in the MS-MS mode compared to other MALDI-TOF systems.
In this paper we demonstrate the application of the MALDI Q-TOF instrument
for high throughput proteomics. A number of modifications have been
made to optimise the system for high throughput proteomics, these include
a MALDI source with a high-density target plate and software that has been
developed for automated data acquisition in both the MS mode and the MS
to MS/MS switching mode. Dedicated processing software has been developed
to fully automate the post acquisition and databank searching. This software
has been optimised to consider the unique nature of the data acquired
from this configuration of instrument.
JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 11, ISSUE 4, DECEMBER 2000 203