FR AB - Science Reference

ABRF 2001 ABSTRACTS
P41-M
Review of gel- and column-based separation technologies for
the identification of yeast proteins using MS/MS.
V.C. Wasinger, G.L. Corthals; Garvan Inst. of Med. Res., 384 Victoria St.,
Sydney, NSW 2010, Australia
Proteome analysis involves the identification and quantitation of expressed
proteins by a given cell type, tissue or organism and most commonly involves
2-dimensional electrophoresis (2-DE) followed by mass spectrometry (MS)
for protein separation and identification. While 2-DE is unequalled in its ability
to separate and resolve thousands of proteins in complex solutions, we
have recently shown that many proteins of biological significance lay beyond
the detection limits of 2-DE. The application of 2-D gels becomes ineffective
when proteins co-migrate to the same grid coordinates, merge to become
one large spot or are not displayed because their concentration is simply too
low for visualisation and subsequent biochemical MS analysis.
Two important biological aspects prevent us from fully exploiting the power
of the 2-DE technology: 1) Firstly, protein expression exists for up to 12
orders of magnitude (in serum). This broad range of expression critically limits
the current 2-DE approach, as at best only 5 orders of magnitude difference
in expression can be displayed; 2) “functional” proteins operate in association
with other proteins; such that research must be directed toward the
analysis of multiply interacting proteins or functional modules that regulate
complex biological networks and pathways.
Alternative fractionation methods such as chromatography exist and allow
proteins/peptides to be separated based on similar (charge, size) or alternative
(affinity, hydrophobicity) properties to 2-DE. Used in series with MS,
these technologies have the potential to fractionate large numbers of proteins
and peptides to enable the comprehensive, comparative and relational analysis
of proteins. Yeast is an ideal choice for this study as both genome, proteome
and transcriptome data is available for evaluation.
A comparison of the separation potential based on protein numbers, protein
classes and properties are described using 2-D gel electrophoresis, size
exclusion and cation exchange chromatography followed by protein identification
using ESI-MS/MS.
P43-S
Characterization of differential protein expression between wild
type and Rce1 knockout mouse embryonic fibroblasts using
2-D SDS PAGE and MALDI TOF mass spectrometry.
S.C. Hall1, D. Smith-Beckerman2, M. Lobo2, S.G. Young3; 1Applied
Biosystems, 850 Lincoln Centre Drive, Foster City, CA 94404,
2San Francisco State Univ., 3UCSF
Ras proteins play an important role in transmitting growth signals from membrane
receptors to the nucleus, triggering the transcription of genes involved
in cellular proliferation. Ras proteins are membrane bound, guanine
nucleotide-binding proteins with GTP-ase activity. Many human cancers contain
mutationally activated Ras proteins that transmit growth signals in an
uncontrolled manner, triggering neoplastic transformation and uncontrolled
cellular proliferation. The proper intracellular location of the Ras proteins
depends on a series of posttranslational modifications: isoprenylation of a Cterminal
cysteine, endoproteolytic release of the carboxyl-terminal three
amino acids, and methyl esterification of the carboxyl-terminal isoprenylcysteine.
Each of these processing steps represents a potential target for cancer
therapy. The endoproteolytic cleavage step is carried out by the product
of the Rce1 gene. Recently, the Rce1 gene was inactivated in mice, completely
blocking the endoproteolytic processing of the Ras proteins. We used
2D-PAGE to assess differences in protein expression in primary embryonic
fibroblasts from wild-type and Rce1 knockout mice. First, we wanted to
detect substrates for Rce1. Altered electrophoretic mobility of individual protein
spots on the gel indicated the possibility of aberrant post-translational
processing. Second, we wanted to detect proteins whose expression level
was affected by the Rce1 knockout mutation. MALDI TOF mass spectrometry
was used to generate peptide mass fingerprints to identify several proteins
having identical molecular weight, pI, and relative abundance in both wildtype
and Rce1 knockout mice. Additional confirmation of the identities of
these proteins was obtained by performing post-source decay analysis on
selected tryptic peptides. It was important to identify these “marker” proteins
as they will be used as migration reference points when comparing future
2D-gel separations. Furthermore, they permitted optimization of protocols for
the MS analysis of Ras proteins from wild-type and Rce1 knockout fibroblasts.
POSTER ABSTRACTS
198 JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 11, ISSUE 4, DECEMBER 2000
P42-T
Quantitative analysis of tumor antigens by mass spectrometry.
A. Kishiyama, D. Arnott; Genentech, 1 DNA Way, South San Francisco,
CA 94080
Both genomics and proteomics are useful for comparing gene expression
patterns. The discovery of receptors overexpressed on tumor cells has lead
to effective cancer therapies, for example. Techniques such as DNA arrays
can be used to measure mRNA levels with great sensitivity and speed. More
detailed information can be obtained through 2D gels, western blotting, and
immunohistochemistry, but these protein-based approaches are time-consuming,
prone to biases, or require generating antibodies. A method has
therefore been developed, called the mass western experiment in analogy to
the western blot, to better bridge genomics and proteomics.
In this experiment, a variation on the ICAT experiment described by Gygi et
al. (Nature Biotech. 1999 v.17 p.994), specific proteins (such as those found
to be of interest from DNA array experiments) are detected and compared
between samples. Proteins extracted from two samples are labeled with a
custom ICAT reagent. The samples are mixed, digested, and the labeled peptides
collected. LC-MS/MS is performed on an ion trap instrument; anticipated
tryptic peptides from the protein of interest are continuously subjected to
CID. Heavy and light ICAT-labeled peptides are simultaneously trapped and
fragmented. Both identification and quantitation is thus obtained in one
experiment.
This approach has been validated by the comparison of cell lines expressing
known tumor antigens in different amounts. Overexpression of the
receptor Her-2 in breast cancer cell lines was shown, and by factors in
agreement with other measurements. Other potential tumor antigens have
likewise been detected.
P44-M
Intelligent data acquisition and automated sample analysis via
orthogonal MALDI- QqTOF, a new tool for protein identification.
C.M. Lock; MDS-Sciex, 71 Four Valley Drive, Concord, Ontario
L4K 4V8, Canada
The application of a novel UV-MALDI ionisation source coupled to an
Applied Biosystems/MDS-Sciex QSTAR Pulsar QqTof mass spectrometer for
protein sequencing and identification is described. The coupling of these two
devices enables collision induced dissociation spectra of singly charged
MADLI ions to be generated, with all the associated QqTof benefits of high
mass accuracy and resolution. The inherent pulsed nature of the o-MALDI
source is converted into a pseudo continuous beam of ions by collisional
cooling in the Q0 region. The o-MALDI source is completely decoupled from
and has no influence on the orthogonal Tof analyser.
High mass accuracy and resolution is thus maintained simultaneously over
the full mass range when switching between MS and MS/MS modes as
opposed to conventional MALDI post source decay experiments.
The application of the technique to the analysis of low femtomole unseparated
protein tryptic digests is demonstrated using an automated data acquisition
approach. The software developed enables the intelligent acquisition
of data from sample plates with minimal user intervention.
The high speed data acquisition capabilities of the o-MALDI source in combination
with the high performance of the QqTof offers unique possibilities
for rapid identification of proteins. Rapid analysis and identification of proteins
via a peptide-mass fingerprinting approach and MS/MS sequence information
will be shown.