Clinical OMICs - Issue 3 - (Page 26)
Clinical OMICs CASE STUDY
A Global Phosphorylation Assay
Ian Pike and Emma Lahert
T
he use of targeted agents
against key signaling kinases is
transforming cancer treatment.
Drugs such as Herceptin and Zelboraf
have increased progression-free survival in breast cancer and melanoma
respectively and are far from the only
examples. However, the complexity
of signaling pathway networks allows
tumor cells to adapt under monotherapy using alternative pathways
to maintain a proliferative phenotype.
Such resistance mechanisms may
be inherently present at the onset
of treatment (de novo resistance) or
may develop in response to the treatment (acquired resistance). This has
led researchers to call for the use of
more comprehensive analysis of signaling pathways to identify resistance
pathways, and to then use this knowledge to select precise combinations
of drugs matched to the individual
tumor profile1,2.
Regulation of pathway activity is
achieved through precisely timed
IAN PIKE is chief operating officer
(ian.pike@proteomics.com)
and EMMA LAHERT is product
manager at Proteome Sciences.
(emma.lahert@proteomics.com)
Website: www.proteomics.com.
26
Clinical OMICs May 15, 2014
gene expression overlain with the
more subtle regulation of posttranslational modifications such as
phosphorylation, glycosylation, and
ubiquitination. While genomic tools
such as RNAseq and next-generation
sequencing may demonstrate gross
changes in signaling networks, only
a comprehensive analysis of protein
post-translational modifications,
particularly phosphorylation, across
thousands of pathway proteins, can
provide the required biological information to enable selection of the
most appropriate drug combination
and switching of therapies.
Current technologies for measuring
protein phosphorylation are based
on site-specific antibodies and rely on
Western blotting, which is labor intensive, or reverse phosphoproteomic
arrays, which have issues of cross-talk.
These techniques are particularly limited in differentiating between close
protein homologs (e.g., Erk1 and Erk
2) or occupancy at adjacent or closely
situated phosphorylation sites. Furthermore, only those proteins/sites
for which high-quality antibodies
are available can be interrogated.
To overcome these limitations, Proteome Sciences has combined state
of the art developments in mass spec-
trometry, chromatography, and isobaric labeling to develop SysQuant, a
global phosphoproteomic workflow
that monitors thousands of molecular
switches regulating hundreds of signaling pathways in up to ten disease
tissue biopsies in a single experiment.
The SysQuant Workflow
SysQuant (Figure 1) requires around
100 mg of fresh or frozen tissue or cultured cells, equivalent to a core needle biopsy or 2 x 15 cm2 dishes, from
which the total proteome is extracted.
Following tryptic digestion and isobaric TMT labeling, up to 10 samples
can be combined into a single analytical mixture for subsequent fractionation and quantification by mass
spectrometry. The analytical mixture
is first fractionated by strong cation
exchange (SCX) followed by selective enrichment of phosphopeptides
(IMAC and TiO2) prior to uHPLC-MS/
MS analysis (Easy-nLC 1000 coupled
to Orbitrap Fusion Tribrid-both
Thermo Scientific).
Figure 1
One set of SCX fractions is analyzed
directly to monitor total protein levels based on nonmodified peptides.
www.clinicalomics.com
http://www.proteomics.com/images/Fig_1-SQ-Workflow.png
http://www.proteomics.com
http://www.clinicalomics.com
Table of Contents for the Digital Edition of Clinical OMICs - Issue 3
Contents
Clinical OMICs - Issue 3
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