& Reports A>Z
HUPO and Cancer Application Reports
Biotech Support Group described a panel of protein biomarkers dysregulated in cancer at the recent HUPO World Congress, that took place September 17-21 in Dublin, Ireland. This research shows that there are three common pathways which change in the bloodstream regardless of the primary tumor, stage, metastatic disease, or tumor burden. Five different cancers – lung, breast, pancreatic, lymph and ovary, were profiled along with normal/healthy individuals of approximate age and matched sex for comparison.
Cancer Proteomics Application Reports
Poster Report Describes Loss of Functional Serpin Activity In Cancer Patient Blood
Dr. Ingrid Verhamme from Vanderbilt University presented a poster, co-authored and in collaboration with Biotech Support Group, describing the loss of functional activity involving 2 major serum proteins, and the potential consequences of such loss in the progression of cancer. The poster was presented at The Serpins2019 Conference, September 19-22, 2019 in Sevilla, Spain, and entitled “Loss of Functional Alpha-1-Antitrypsin and Heparin Cofactor II in Inflammation and Cancer”. Authors were: Ingrid M. Verhamme, Vanderbilt University Medical Center; Nashville TN, Swapan Roy, Sowmya Avadhani, Matthew Kuruc, Biotech Support Group LLC, Monmouth Junction NJ.
Highlighting the importance of this poster report, Dr. Verhamme states that “Our hypothesis is that there may be a common host systemic response to many forms of cancer, regardless of primary tumor, stage or development of metastatic disease. This response is proposed to involve interconnected pathways attributable to thrombo-inflammation and innate immunity. All these pathways are activated by proteolysis and regulated by protease inhibition. So it is therefore likely that tumorigenesis can systemically be characterized by chronic exhaustion of inhibitory active serpins, and resulting increased protease activity. Our results support such a net decrease in inhibitory active Serpin D1, otherwise known as Heparin Cofactor II. We believe this is a very important discovery as it brings to light a potential new cancer therapeutic strategy, to selectively inhibit Thrombin in the extravascular space, as 60% of Heparin Cofactor II is extra-vascular.”
The poster describes that in various cancer types, blood serum levels of serpins have been reported as altered. Significantly however, is that in these reports, concentrations were measured by immunological methods (ELISA) rather than by functional activity. This can lead to egregiously misleading interpretation of a host’s systemic response to cancer, as the immunological assay measures in aggregate both the intact and cleaved serpin forms. In contrast to these methods, we demonstrate that with albumin depleted (AlbuVoid™) serum samples, combined with LC-MS/MS, there is potential of distinguishing between active and cleaved subpopulations of Serpins. Cleaved serpins are the product of the substrate pathway in the bifurcated serpin suicidal mechanism, which can become more pronounced due to serpin mutations or changes in the micro-environment.
This study considers that micro-environmental changes in tumors are sufficient to distort the ratio of cleaved serpin forms from intact serpin forms, and that these ratios are sufficiently differentiated so as to be reportable in the general blood circulation. Serpins A1 (Alpha-1-Antitrypsin) and D1 (Heparin Cofactor II) primarily regulate the activities of the inflammatory proteases elastase and thrombin respectively. Using functional enzyme assays, preliminary results correlate to LC-MS/MS results, confirming a significant increase in the cleaved/total ratio in cancer sera, corresponding to a net decrease in inhibitory active serpin, relative to a normal/healthy status.
Innate immunity adaptations are determined by fluid mechanics, so mediators of coagulation in the tumor vasculature are differentially regulated from those of the general circulation. As a result, the permissive conditioning necessary for metastasis is derived from the micro-vasculature at the primary site; loss of Heparin Cofactor II (Serpin D1) function being one important contributor.
Swapan Roy, Ph.D., President and Founder of Biotech Support Group, concurs stating “These are very exciting developments as methods to monitor the influence of SERPINs is a fundamentally new way to think about the microenvironment componentry of cancer. Through our academic collaborators, we continue to generate evidence that there is a fundamental dysregulation of protease inhibition in tumors that supports the host’s stromal conditioning. With that re-education comes the allowance for cancer to survive and metastasize. Most importantly, these same systemic responses which are derived from tumor tissue vasculature now can be monitored using liquid biopsies. From this, new avenues for diagnosis, personalized medicine, and therapeutic modalities are possible. Our next goal is for rigorous investigations to characterize progressive disease or response to therapies with partners and collaborators. This will be our next step towards our ultimate goal of validating biomarkers useful in the clinic. We welcome inquiries to commercialize our patent pending biomarkers, called Stroma Liquid Biopsy™, for the many different applications it can serve including early detection, wellness and risk factor monitoring, oncology drug development and personalized therapy decisions.”
To Learn More About Stroma Liquid Biopsy™, download whitepaper at:
For more information on the BSG complete line of Albumin & IgG Removal products, visit: https://www.biotechsupportgroup.com/Articles.asp?ID=451
Whitepaper Describes Stroma Liquid Biopsy™ Cancer Serum Biomarker Panel
Biotech Support Group announced today that it has published a whitepaper describing Stroma Liquid Biopsy™, a pan-cancer biomarker panel of dysregulation derived from blood. The title and download link is:
Stroma Liquid Biopsy™ - Blood-based biomarkers to monitor stromal conditioning in cancer. Published February, 2019.
The whitepaper describes that tumors are more than simply a collection of immortalized cells as the supporting microenvironments or stroma also contributes to pathogenesis. Because of this, tumor characterization cannot be fully characterized solely through the analyses of the tumor cell genome – the current emphasis of liquid biopsy platforms. So because tumors are more than just a mass of proliferating cells, cancer progression must take into consideration the influence of the multiple cell types and networks of proteins dynamically interacting in active tumorigenesis. These are not simply passive bystanders.
The unique significance of the Stroma Liquid Biopsy™ pan-cancer profile is that dysregulation in blood was categorically intertwined with the most rudimentary needs of cancer: space, nutrients and immune evasion. Moreover, the changes within the 13 biomarker panel all occur within an interdependent network of cascading proteolytic events. Because proteolysis is irreversible, all species of life have evolved molecular regulatory systems to control aberrancies. The most distinguished is a protease inhibitory family of regulators known as SERPINs. Although SERPINs circulate in a variety of functional on/off sub-forms, conventionally they are observed and reported in aggregate (i.e., ELISA). As a result, their influence on disease is missed. Using combined LC-MS peptide features and enzyme assay methods that directly assess SERPIN function, rather than antigen presentation, we now have evidence that there is an imbalance of functional SERPIN sub-forms in the cancer population relative to that of a normal population.
Poster Reprint First Presented at New Jersey Cancer Retreat, May 25, 2017.
The poster authors and title are:
Haiyan Zheng(1), Swapan Roy(2), Amenah Soherwardy(1), Seema Rahman(2), Matthew Kuruc(2); (1)Rutgers Center for Integrative Proteomics, Piscataway, NJ; (2)Biotech Support Group LLC, Monmouth Junction, NJ; entitled
In brief, while the landscape of cancer-associated DNA mutations has supported the initial concepts for liquid biopsy, it is now overwhelmingly apparent that throughout cancer progression, there are necessary adaptive microenvironments to support metastatic disease. We now present evidence that some of the essential interactions between stroma and proliferating cells can in part, be monitored through the protein response that tracks into the vascularized tumor and re-proportions the extracellular proteins (serum) found in the general blood circulation. These patent pending proteomic patterns can now be reported as a Stroma Liquid Biopsy.
Poster Reprint First Presented At AACR Annual Meeting 2016 Conference, Held April 17-20, 2016 New Orleans, LA USA.
The Commonality of the Cancer Serum Proteome Phenotype as analyzed by LC-MS/MS, and Its Application to Monitor Dysregulated Wellness Haiyan Zheng1; Caifeng Zhao1; Swapan Roy2; Devjit Roy MD; Amenah Soherwardy2; Ravish Amin2; Matthew Kuruc21Rutgers Center for Integrative Proteomics, Piscataway, NJ; 2Biotech Support Group LLC, Monmouth Junction, NJ
Princeton based Biotech Support Group announced today that it has submitted provisional patents disclosing its data on many common proteins dysregulated in cancer. This breakthrough research shows that most of these biomarkers are believed to be dysregulated regardless of the primary tumor, stage of progression or tumor burden. The study which collaborated with the Rutgers Center for Integrative Proteomics shows the most distinguishable pattern of early dysregulation observable as a cancer serum phenotype to date. This forms the basis of a new commercialization plan featuring:
Variant sub-populations (also known as proteoforms) of a common blood protein – Alpha-1-Antitrypsin, with functional reporting features severely distinguishable between cancer patients and normal/healthy individuals. A measurable serum cancer profile that can be modeled with categorical biomarker proteins taken from inflammation, blood coagulation, tissue remodeling, and glycolysis, as well as new markers of unknown function.
Cancer Biomarker And Wellness Protein Discovery Application Report
Haiyan Zheng1; Caifeng Zhao1; Swapan Roy2; Devjit Roy MD; Amenah Soherwardy2; Ravish Amin2; Matthew Kuruc2 1Rutgers Center for Integrative Proteomics, Piscataway, NJ; 2Biotech Support Group LLC, Monmouth Junction, NJ. Application Report -
The Comparison of the Serum Proteome in Individuals with Cancers versus those without Cancer, and its application to Wellness
Biomarker discovery from genomic analysis of singlular biomarkers or protein panels can be enhanced by doing albumin depletion experiments and on bead digestion. A wellness proteome strategy of over 200 spectrally quantified proteins from healthy/normal or disease cancerous sera of pancreatic, breast, lung patients are obtained. Individuals from pancreatic, breast and lung cancer sera are analyzed by adding a binding buffer. The bead retains serum proteome and albumin is depleted. Subsequently the serum proteins on bead are analyzed by wash, reduction, alkylation, trypsin digestion and are labelled. Spectra from liquid chromatography mass spectrometry (LC-MS/MS) runs and proteins analyzed by Xtandem with protein filters are obtained. An example of a protein is the SERPIN family protease inhibitor. It allows validating quantitative proteomics data, discovering newly annotated serum proteome.
Swapan Roy, Matthew Kuruc. The Functional Subproteomes of Serpin Protease Inhibitors are Now Open for LC-MS Biomarker Discovery. MOJ Proteomics Bioinform 2016, 3(6): 00106
The authors consider that the conformational variants of the unique family of protease inhibitors annotated as SERPINs, are most often underrepresented in proteomic analyses. This limits understanding the complex regulation that this family of proteins presents to the networks within the protease web of interactions. Using bead-based separation provided by the NuGel™ family of proteomic enrichment products - notably AlbuVoid™ & AlbuSorb™, the authors demonstrate their utility to satisfy investigations of serum SERPINs. The authors also suggest their use to develop functional profiles of the SERPIN Proteoform, and how those can establish relationships to disease phenotypes, gene mutations, and deregulated mechanisms.
Featured Application - Functional Proteomics
Sun, Zhenyu, Xiaofeng Chen, Gan Wang, Liang Li, Guofeng Fu, Matthew Kuruc, and Xing Wang. "Identification of functional metabolic biomarkers from lung cancer patient serum using PEP technology." Biomarker Research 4, no. 1 (2016): 1.
Coupled to On-Bead Digestion - Tackling the Challenges of Serum
Proteomics Bioinform 2015, 8:9
cancer research, serum and plasma are especially attractive sample
types as collection of blood is common, simple and only minimally
invasive. Yet serum samples can offer unique challenges in LC-MS
proteomic analyses. The two biggest challenges being: 1) the high
abundance of Albumin accounting for about 50% of the total protein
mass and, 2) proteolytic resistance, in large part due to substantial
amount of glycoprotein, a modification that manifests proteolytic
resistance. In this short report, we describe new methods using a
surface/bead based product, AlbuVoid™, which depletes Albumin
through a negative selection or voidance strategy, retaining the vast
amount of the remaining serum proteome on the bead. We then combine
this novel enrichment, with a direct and seamless integration with
Trypsin digestion, a method conventionally referred to as on-bead
digestion. We evaluated the digestion time as a parameter to identify
whether different sub-populations of peptides and proteins can be
observed by LC-MS analyses. Using 2 different allotted digestion
times - 4 hours, and overnight, each with a singular 3 hour gradient
LC-MS run, between 400-500 total proteins were observed for both
human and rat sera, with overlapping and distinct sub-populations
observable at each digest time. These results support that the
described methods gain efficiencies over other high abundance
depletion and in-solution digestion workflows. We solicit that such
workflows will minimize many of the inconsistencies of proteolytic
hydrolysis for both discovery and quantitative serum proteomic
this short case study, the enrichment of select sub-populations of
proteins is beneficial to systematically analyze protein functions of
a whole enzyme family from an entire proteome. AlbuVoid™ was used
to remove Albumin and enrich the low abundance proteome, noting that
distinguishable activity features are presented from the lung cancer
vs. the normal sera. KinaSorb™ was used to enrich for both a narrow
spectrum substrate profile—Hexokinase activity, and a
broad-spectrum protein kinase activity. The number of observable
features was consistent with such narrow and broad-spectrum
activities. AlbuVoid™enrichment and PEP processing proved suitable
for profiling the functional activities of Hexokinase, Protease and
Alkaline Phosphatase. These enzyme feature profiles are indicative of
the functional diversity that can be generated, annotated and
compared within and between sample phenotypes, using the combined
Removal Application Report.
a foundational NuGel™ platform chemistry, a library of bead
architectures has been created to support proteomic enrichment. These
beads are general non-specific protein adsorbents, or stated another
way - beads with weak affinity or imperfect fit interactions. Two of
our products support Albumin Removal: AlbuSorb™ for selective
binding of Albumin & AlbuVoid™ for negative selection or
voidance of Albumin with consequent enrichment of the remaining serum
proteome on the bead. We now report on adding Immunoglobulin
depletion as an extension to AlbuSorb™. A LC-MS/MS analysis on
human serum revealed between 500-600 total proteins, many of which
are qualitatively and quantitatively biased to sub-proteomes either
depleted of Immunoglobulins, or not depleted of Immunoglobulins.
AlbuSorb™ PLUS designates and distinguishes AlbuSorb™ without
immunoglobulin depletion, from AlbuSorb™ with Immunoglobulin
depletion. Here we compare proteomic data derived from AlbuSorb™,
AlbuSorb™ PLUS (which includes Immunoglobulin depletion), and
Removal Reference Applications
Two NuGel™ based products support
- Lipid Removal and Cell Response Applications
“omics” revolution demanded new and different sample prep
separations that were not efficiently performed by conventional
technologies. While effective for many applications, these tools were
not efficient for “omics” sample preparation, as
throughput, economy and simplicity are especially required.
Furthermore, these same separation tools often denatured proteins
which limited there use in applications which demanded the
measurement of function, structure or bio-activity. For these
reasons, BSG has been dedicated to create new methods and
applications to drive efficient workflows and better data quality for
all proteomic and biomarker analyses. Of special importance is the
value created when certain families of biomolecules can be evaluated
with respect to cell response and viability. For example,
extracellular vesicles (EVs) substantially influence cultured cell
behavior. While all of our products serve cell response applications,
we report here an extensive list of applications in this area for
Cleanascite™. Cleanascite™ is derived through a
proprietary formulation of metallic oxide derivatives. However,unlike
other metallic oxides, Cleanascite™ does not have significant
protein binding, making its selectivity profile for lipids un-rivaled
in the bio-research products industry. As a result, it is ideal to
clear lipid-associated matrix effects - including extracellular
vesicles, which may influence cell response assays.
download whitepaper entitled “Cleanascite™ - Lipid
Removal and Cell Response Applications"
is derived through a proprietary formulation of metallic oxide
derivatives. Unlike other metallic oxides, Cleanascite™ does not
have significant protein binding making its selectivity profile for
lipids unique in the bio-research products industry.
The applications and references for use of Cleanascite™ are described in this downloadable report.
& Chemical Proteomics
post-translational modification, and non-covalent binding factors all
play a role in fine tuning polypeptide sequence to final function.
Because of this phenomena, populations of proteins annotated to the
same sequence nevertheless can display multiple functions within
tissues and disease states. Likewise, the same or similar function
may be presented by multiple sequences, with subcellular control
mechanisms regulating functional diversity. As a consequence,
strictly abundance based biomarkers may lack the necessary dynamic
range and greater specificity provided by functional based
biomarkers, to define the phenotype. Thus, Functional Proteomic
techniques such as described here, support a top-down proteomic
strategy starting with functional annotation of the structurally
intact protein, and ending with sequence and structural annotation.
of ancient metazoan macromolecular complexes
Nature Volume:525, Pages:339–344 Date published:(17 September
of BSG products,
NRicher™ 6 and HemogloBind™, were able to
contribute to this rigorous examination of protein complexes. When
our products were used as a pretreatment step in the overall
workflow, about twice the number of observations and annotations
became possible. This further validates that the sub-proteome bias
NRicher™ 6 can simplify complex proteomes into
less complex sub-proteomes with efficiencies suitable for deep
functional proteome characterization. Furthermore, this study
demonstrated the importance of a key feature implicit to all of our
products; that is the maintenance of functional and structural
integrity after separations. Without that particular feature, these
additional observations would not have been possible.
efforts in proteomics seek to identify and sequence annotate the
proteome by LC-MS/MS analyses of peptides derived through proteolytic
processing of the parent proteome. However, little attention has been
paid to functional annotation. Yet functional annotation is crucial
as the landscape of protein conformations is highly variable, and
each conformation may contribute to its own unique activity. To
overcome inefficiencies in functional annotation, a new method
combining the enrichment attributes of Biotech Support Group
products, along with the PEP platform developed by Array Bridge (St.
Louis, MO) is described. The PEP technology is a top-down method
which starts with functional annotation and ends with sequence and
structure information. It is based on a modified 2D Gel
Electrophoresis to separate proteomes, electro-eluting from the PEP
plate and systematically measuring enzyme activities from 384
as a Marker
and Toiletries Vol. 129, No. 7, September 2014.
is general agreement on elastin’s role in skin health, aging and
appearance, but no method has yet elucidated how it degrades with
aging when challenged by sun exposure or other means.This paper
reviews two inconsistent theories in the literature, degradation and
buildup, and highlights potential methods to marry the two. It also
suggests proteomics as a method for further investigation.
platform was applied to these publications
Profiling of Phosphodiesterases
Journal of Proteomics. Volume 2012, Article ID 515372, 8
surface chemistries reported here as SeraFILE in this article form
the basis of the product NRicher™ 6.
proteomic profiling can help identify targets for disease diagnosis
and therapy. Available methods are limited by the inability to
profile many functional properties measured by enzymes kinetics. The
functional proteomic profiling approach proposed here seeks to
overcome such limitations. It begins with surface-based proteome
separations of tissue/cell extracts, using SeraFILE, a proprietary
protein separations platform. Enzyme kinetic properties of resulting
subproteomes are then characterized, and the data integrated into
proteomic profiles. As a model, SeraFILE-derived subproteomes of
cyclic nucleotide-hydrolyzing phosphodiesterases (PDEs) from bovine
brain homogenate (BBH) and rat brain homogenate (RBH) were
characterized for cAMP hydrolysis activity in the presence (challenge
condition) and absence of cGMP. Functional profiles of RBH and BBH
were compiled from the enzyme activity response to the challenge
condition in each of the respective subproteomes. Intersample
analysis showed that comparable profiles differed in only a few data
points, and that distinctive subproteomes can be generated from
comparable tissue samples from different animals. These results
demonstrate that the proposed methods provide a means to simplify
intersample differences, and to localize proteins attributable to
sample-specific kinetic responses. It can be potentially applied for
disease and non-disease sample comparison in biomarker discovery and
drug discovery profiling.
reprint first presented at US HUPO April 6-8, 2014.
Removal Reference Applications
BSG products support Hemoglobin Removal applications:
& NuGel™ HemogloBind™ for selective binding of Hemoglobin &
for negative selection or voidance of Hemoglobin with consequent
enrichment of the remaining erythrocyte proteome on the bead
are many DNA, RNA, and nucleic acid preparation products that serve
the market well for most applications. Most of these products in some
way utilize a bind/wash/elute strategy adopting surface reagents such
as silica & metallic oxides and similar solid-phase
chemistries.However, there still remain situations when these
products are not optimal. This Handbook serves to guide users in the
use of ProCipitate™ when the “other kits don’t fit”.
ProCipitate™ strategy is opposite to common prep strategies, as
instead of binding nucleic acids, the protein is efficiently depleted
with no interaction with the soluble nucleic acids. ProCipitate™ is
offered in a variety of kits, providing all necessary buffers and
accessories for different applications: ProPrep™ Genomic 96/100 for
high throughput whole blood DNA; ProPrep™ BAC Mini for large insert