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Stroma Liquid Biopsy™ Whitepaper-January 2022

This whitepaper highlights the importance of the systemic inflammatory response to the presence of cancer anywhere in the body. It also describes how the vast circuitry of cascading proteolytic events, requires suitable regulation in order to resolve innate immunity and accommodate the adaptive T-cell response in cancer. From this, new strategies for more durable therapeutic efficacy will be uncovered.

Biotech Support Group and Leiden University Medical Center Report on a Gene Signature Ratio derived from Stroma Liquid Biopsy™ that Predicts Survival in Colon Cancer

Ravensbergen, Cor J., et al. "
The Stroma Liquid Biopsy Panel Contains a Stromal-Epithelial Gene Signature Ratio That Is Associated with the Histologic Tumor-Stroma Ratio and Predicts Survival in Colon Cancer." Cancers 14.1 (2022): 163.

Liquid biopsy has emerged as a novel approach to tumor characterization, offering advantages in sample accessibility and tissue heterogeneity. However, as mutational analysis predominates, the tumor microenvironment has largely remained unacknowledged in liquid biopsy research. The Stroma Liquid Biopsy™ (SLB) proteomics panel comprises a set of 13 proteins from interconnected stromal pathways (i.e., coagulation, complement, acute phase inflammation) and is believed to capture a plasma proteomic blueprint indicative of a deranged systemic response in cancer. As such, it encompasses the importance of the tumor microenvironment (TME) compartment in liquid biopsy. Within similar context, the histologic tumor-stroma ratio (TSR), a stroma-derived biomarker developed by LUMC, has been validated as an independent predictor of patient survival in various primary tumor types. The current work provides an explorative gene transcriptomic characterization of the SLB proteomics panel in colon carcinoma by integrating single-cell and bulk transcriptomics data from publicly available repositories.

A Preliminary Investigation Using Targeted LC-MS Proteomic Methods Demonstrates Unique Serum Profiles of Hospitalized SARS-CoV-2 Patients

Douglas D Fraser 1; Haiyan Zheng 2; Swapan Roy 3; Matt Kuruc 3; Amenah Soherwardy 2; Devjit Roy 4; Sowmya Avadhani 3

1Lawson Research Institute, London, Ontario; 2Rutgers University Proteomics Center, Piscataway, New Jersey; 3Biotech Support Group LLC, Monmouth Junction, NJ; 4Nathan Littauer Hospital, Gloversville, New York Corresponding Author : mkuruc@biotechsupportgroup.com

Brief description: For serum samples, targeted LC-MS is challenging, mainly due to the presence of highly abundant proteins. So it becomes critical to pair target peptides to sample depletion methods to best establish differentiated profiles between samples. In this preliminary investigation, we use unique methods described here to characterize the functionality of the innate immune response in hospitalized Covid-19 patients, more precisely than current methods.

High-throughput, Automated INTip™ SPE Combined with AlbuSorb™ Products for Efficient Albumin and Albumin/IgG Depletion

Matthew Fitts 1; William E. Kemnitzer 1; Paul Meeh 1; Matt Kuruc 2; Swapan Roy 2; Sowmya Avadhani 2

1DPX Technologies, Columbia, SC; 2Biotech Support Group LLC, Monmouth Junction, NJ Corresponding Author: mkuruc@biotechsupportgroup.com

Brief description: We report here new methods to incorporate beads for depletion of Albumin, IgG and Hemoglobin, into pipette tips known as XTRaction tips or XTR tips. The XTR tip format improves ease of use and scalability to process multiple samples in parallel, utilizing 96-well plates and automated liquid handlers. The incorporated BSG beads are consumable, and not derived from immuno-affinity.

These two posters highlight the value of collaborative partners jointly working to commercialize new methods and biomarkers using novel proteomic sample prep products. The first demonstrates how we can adapt LC-MS to study the functionality of a most important regulating family of proteins - the Serpins, as they serve as central control within the innate immune response. The second demonstrates our commitment to high-throughput and automation for clinical proteomics. It is very exciting to share these developments with a dedicated group at the forefront of mass spectrometry research”, states Dr. Swapan Roy, Ph.D., President and Founder of Biotech Support Group.

Journal Publications

Albumin Removal Kits

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.

Zheng et al. AlbuVoid™ Coupled to On-Bead Digestion - Tackling the Challenges of Serum Proteomics . J Proteomics Bioinform 2015, 8:9

For 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 applications.

Xing Wang, Michael Davies, Swapan Roy and Matthew Kuruc. Bead Based Proteome Enrichment Enhances Features of the Protein Elution Plate (PEP) for Functional Proteomic Profiling . Proteomes 2015, 3(4), 454-466 . doi: 10.3390/proteomes3040454

In 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 methods.

Application Reports

Albumin Removal Application Report.

AlbuSorb™ Product Extension combines Albumin and Immunoglobulin Depletion in a Consumable Format

From 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 AlbuVoid™.

New Proteomic Workflows Combine Albumin Depletion and On-Bead Digestion, for Quantitative Cancer Serum
Poster reprint first presented at 14th Human Proteome Organization World Congress (HUPO 2015), held September 27 – 30, 2015 Vancouver, British Columbia, Canada.

Improved proteomic enrichment and workflow strategies
Poster reprint first presented at US HUPO April 6-8, 2014.

Albumin Removal Reference Applications
Two NuGel™ based 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.
The applications and references for use of these products are described in this downloadable report.


Cleanascite™ - Lipid Removal and Cell Response Applications

The “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.

To download whitepaper entitled “Cleanascite™ - Lipid Removal and Cell Response Applications"

Visit: http://www.biotechsupportgroup.com/v/vspfiles/templates/257/pdf/CleanasciteCellResponseReferenceApplications121218.pdf

Cleanascite™ 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.

Functional & Chemical Proteomics

The Functional & Chemical Proteomics Handbook

Mutations, 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.

Journal Publications

C Wan, B Borgeson , S Phanse , F Tu, K Drew , G Clark , et al. Panorama of ancient metazoan macromolecular complexes . Nature Volume:525, Pages:339–344 Date published:(17 September 2015). doi:10.1038/nature14877

Two 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 characteristics of 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.

Wang, X. et al. Discovery of Functional Serum Markers . ClinicalOMICS February 2015.

Most 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 microwell plates.

Elastin as a Marker . Cosmetics and Toiletries Vol. 129, No. 7, September 2014.

There 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.

NuGel surface platform was applied to these publications
Functional Proteomic Profiling of Phosphodiesterases

International Journal of Proteomics. Volume 2012, Article ID 515372, 8 pages. doi:10.1155/2012/515372.

The surface chemistries reported here as SeraFILE in this article form the basis of the product NRicher™ 6.

Functional 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.

NuGel™ NRicher™ Mx: Compound-centric Displacement Proteomics – An advantaged method to survey small molecule-protein interactions

Poster reprint first presented at US HUPO April 6-8, 2014.

For HemogloBind & HemoVoid

Review Reports

Hemoglobin Removal Reference Applications

Three BSG products support Hemoglobin Removal applications:

  • HemogloBind™ & NuGel™ HemogloBind™ for selective binding of Hemoglobin &

  • HemoVoid™ for negative selection or voidance of Hemoglobin with consequent enrichment of the remaining erythrocyte proteome on the bead

The applications and references for use of these products are described in this downloadable report.


Superior Substitute to Phenol/Chloroform for DNA Isolation & Protein Binding Handbook of applications, strategies, and potential new directions

There 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”.

The 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 plasmid preps.