SeraFILE™SeraFILE™ - A Biomarker and Drug Discovery Engine
SeraFILE™ (patent pending)
is a surface library for sorting proteins for differential analysis. The SeraFILE™ surface library is not
based on conventional liquid chromatography stationary phases. Whereas conventional LC (green) suffers from
a heterogeneous mix of binding energies, and Affinity (red) has exceedingly high binding energy, SeraFILE™
promotes weak, homogeneous binding - ideal for proteomic investigation. Selectivity modulation comes from the
structural morphology and spatial distribution of immobilized electrolytes, thus generating protein pools
with both differential constituents and activity states.
SeraFILE™
The SeraFILE™ surface library provides:
- A modality that is open-ended and industrially productive.
- Reduced protein complexity with subsequent maintenance of native, biologically functional conformations.
- New profiling techniques which generate signatures across a multiplicity of sub-proteomes and interrogations.
- A means to characterize enzyme regulation and related functional sub-states from disease.
- Discovery strategies that enrich catalytic activity and directly couple to drug development.
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Low Abundance Protein Enrichment
The SeraFILE™ surface library can be used in serial strategies for enrichment of low abundance proteins. Like peeling an onion, one surface can void one or more high abundance proteins (see serum example), and concentrate the low abundance proteins. If necessary, more than one surface can be used in series, to provide maximal enrichment and resolution of proteins from gel electrophoresis visualization.
The SeraFILE™ surface library can be used in serial strategies for enrichment of low abundance proteins. Like peeling an onion, one surface can void one or more high abundance proteins (see serum example), and concentrate the low abundance proteins. If necessary, more than one surface can be used in series, to provide maximal enrichment and resolution of proteins from gel electrophoresis visualization.
Secondary Interrogation
An immediate and direct hand-off simplifies secondary interrogation. Available methods include conventional analytical options (such as 1 and 2 Dimensional Electrophoresis, & structural assays), outsourced techniques (i.e., Mass Spec) and proprietary functional metrics, as highlighted in yellow.
An immediate and direct hand-off simplifies secondary interrogation. Available methods include conventional analytical options (such as 1 and 2 Dimensional Electrophoresis, & structural assays), outsourced techniques (i.e., Mass Spec) and proprietary functional metrics, as highlighted in yellow.
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These measurements collectively provide a molecular profile (or "signature") of the
sample forming a knowledge-base. Comparison algorithms applied to the knowledge-base identify patterns
associated with disease. The goal is, through a critical number of clinically defined samples, to correlate
proteins individually or as a nexus to disease.
Along with conventional proteomic analyses, SeraFILE™ participates in two unique discovery
avenues especially for drug development:
- Rational Proteome Prospecting™, an iterative strategy to isolate biomarkers of interest, and
- Conformerics™, an induction of enzymatic sub-states (or conformers) which can be profiled, isolated and developed into targets for drug screening assays.
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The discovery of protein biomarkers is severely hindered by the high abundance proteins
which are uninteresting and can mask the presence of the putative marker. Due to the increased
sensitivity of low molecular weight component analysis, the presence of a protein can oftentimes be
determined by measurement of its derivative products, even when no protein marker is evident. SeraFILE™
provides such a rational tiered method for isolating the causative 'needle in the haystack' protein(s).
SeraFILE™ can deliver multi-faceted proteomic signatures combining elements of protein abundance as well
as functional activities.
These elements can be used to "score" the various pools for enrichment of desirable
activity. Each sub-proteome is scored - by bioassay, HPLC, NMR, or mass spectrometry, relative to its total
protein content. The high-score fractions are selectively applied to additional tiers of enrichment; each
tier producing successively greater levels of purification. Because functional activity is maintained in all
pools, Rational Proteome Prospecting™ can be applied to any measurable metabolite or byproduct. Also, through
pool recombination, Rational Proteome Prospecting™ techniques can be applied to discover important enzyme
regulating factors..
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Ubiquitin/Proteasome Pathway (UPP) Characterization.
Background.The 20S proteasome forms the catalytic core of the UPP complex, responsible for proteolytic activity. The 19S regulatory complex associates with either or both ends of the 20S complex. The 19S regulator is involved in the recognition, binding and de-ubiquitinylation of ubiquitinylated proteins tagged for destruction, and stimulates its proteolytic activity.
Results. Western Blot analysis of SeraFILE™ -derived yeast sub-proteomes. Ub(n) regions visualize proteins conjugated to Ubiquitin; Numbers 1-11 correspond to the SeraFILE sub-proteomes; RPT1 regions are indicative of 19S; Bottom Line numbers are measurements of specific proteolytic activity of the 20S particle. Control Proteolytic Activity (without SeraFILE™ treatment) was 82 Units.
Low Abundance Protein Enrichment
The SeraFILE™ surface library can be used in serial strategies for enrichment of low abundance proteins. Like peeling an onion, one surface can void one or more high abundance proteins (see serum example), and concentrate the low abundance proteins. If necessary, more than one surface can be used in series, to provide maximal enrichment and resolution of proteins from gel electrophoresis visualization.
The SeraFILE™ surface library can be used in serial strategies for enrichment of low abundance proteins. Like peeling an onion, one surface can void one or more high abundance proteins (see serum example), and concentrate the low abundance proteins. If necessary, more than one surface can be used in series, to provide maximal enrichment and resolution of proteins from gel electrophoresis visualization.
Conformerics™
SeraFILE™ generates alternate conformers in vitro. Such conformers may arise from discrete docking changes in the polypeptide chain, or through dissociation of positive or negative regulating factors, or some combination of the two. In all cases, the capability to exploit enzymatic variants and sub-states provides a new drug development strategy. Rather than blocking the activation of inactive conformers- the conventional drug discovery route, the ProFACT paradigm would promote deactivation from active states.
SeraFILE™ generates alternate conformers in vitro. Such conformers may arise from discrete docking changes in the polypeptide chain, or through dissociation of positive or negative regulating factors, or some combination of the two. In all cases, the capability to exploit enzymatic variants and sub-states provides a new drug development strategy. Rather than blocking the activation of inactive conformers- the conventional drug discovery route, the ProFACT paradigm would promote deactivation from active states.
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The presumed advantages are that the active conformer is more specific to disease than the
inactive conformer, and that its lower stoichiometry would translate to lower dosages and lower toxicity.
Unique and proprietary to ProFACT, these combined deliverables will provide an exceptional new approach to
accelerate drugs to market. Rational Proteome Prospecting ™ can be used to isolate or purify important drug
targets. Conformerics™ will integrate these targets within drug screening assays.
A Functional Difference
Investigations into the Ubiquitin/Proteasome Pathway have revealed distinct functional proteomic signatures in cancer and normal tissue. Several SeraFILE™-induced subproteomes demonstrated conformationally distinct variants or conformers - notated in red below. Evidence of soluble regulatory factors as well as conformers resistant to inhibitors has been uncovered.
Investigations into the Ubiquitin/Proteasome Pathway have revealed distinct functional proteomic signatures in cancer and normal tissue. Several SeraFILE™-induced subproteomes demonstrated conformationally distinct variants or conformers - notated in red below. Evidence of soluble regulatory factors as well as conformers resistant to inhibitors has been uncovered.
SeraFILE™-derived enzymatic activity profiles may provide a means to detect
disease-specific differences and conformers to drug screens.
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Proteasome chymotryptic activity increased significantly in cancer samples in most
sub-fractions, for breast (A) and esophageal cancer (data not shown), compared to normal adjacent tissue
(the control). Red arrows indicate altered proteasome activity in a few notable sub-proteomes compared to
control extracts (for instance flow-through fractions from matrix 1 and 11; B).
Altered proteasome activity in certain sub-fractions may have clinical relevance because 1)
disease-specific inhibitors (i.e. potential drug targets) may be identified in other fractions and 2)
activity profiles may function as disease-specific biomarkers.
SeraFILE™ Service Deliverables
SeraFILE™ can be advantageous in routine proteomic investigations using 1DE/2DE/Mass Spec approaches, or through ProFACT's proprietary menu of services. Deliverables are client driven and can include:
SeraFILE™ can be advantageous in routine proteomic investigations using 1DE/2DE/Mass Spec approaches, or through ProFACT's proprietary menu of services. Deliverables are client driven and can include:
- Differential Sub-proteome pools
- Molecular Disease Signatures
- Bioassay Profiles
- Reproducible Quantitative Abundance Profiles
- Functional Pathway Characterization
- Rational Proteome Prospecting™
- Conformerics™ Bioactive Target/Drug Assays