The 4 common features of our sample prep products, known as the BSG Advantage, are highlighted in a selection of journal references

The 4 common features of our sample prep products, known as the BSG Advantage, are highlighted in a selection of journal references.

BSG Advantage

All of our proteomic sample prep products have these 4 common features and collective advantages:

Consumable

Cost-effective, not derived from biologicals

Not derived from immuno-affinity, no regeneration, no specialized instruments of HPLC, compatible with high-throughput and automation

Enrichment/Depletion

Diverse strategies, species agnostic

Depletion of high abundance, or enrichment of low abundance. product line covers both strategies for Albumin & IgG removal, and Hemoglobin Removal

On-Bead Digestion

Efficient workflows, quality LC-MS data
Pioneered Bead-Assisted Sample Prep (BASP™), new efficiencies for LC-MS proteomics of bead enriched sub-proteomes

Functional Integrity

Maintained throughout separations
Supports functional, chemical and top-down proteomics, cell reponse

Background

Biotech Support Group (BSG) is a leading manufacturer and supplier of sample preparation products. We are dedicated to create new methods and applications to drive efficient workflows and better data quality for all proteomic and biomarker analyses. For this, we focus all of our products and methods to provide certain advantages.

The Outcomes

BSG’s Collective Advantages are reported in hundreds of journal references. For more than 10 years, BSG has been the innovator in engineered beads with differential physicochemical properties for sub-proteome separations, first reported in the 2015 Nature article referenced below. BSG also pioneered on-bead digestion of bead-enriched sub-proteomes. In this case study we highlight representative journal publications whereby the BSG advantage uniquely supports the research study goal. These investigations cover a wide range of diseases, including cancer, neurodegenerative, Covid-19, malaria, and cardiovascular.

BSG Advantage: Consumable ● Species Agnostic

Phillippi, Danielle T., et al. "Inhaled diesel exhaust particles result in microbiome-related systemic inflammation and altered cardiovascular disease biomarkers in C57Bl/6 male mice." Particle and fibre toxicology 19.1 (2022): 1-29.

Exposure to particulate matter (PM) is a known mediator of inflammation and CVD. However, the role of inhaled traffic-generated PM on the gut microbiome and its corresponding systemic effects are not well-characterized. Thus, the study investigated exposure to inhaled diesel exhaust particles (DEP) on mice. To determine systemic inflammation, following HemogloBind™ treatment, plasma concentrations of 20 Cytokines were measured using a Mouse Cytokine/Chemokine Magnetic Bead Panel; the article stating “HemogloBind was added to plasma samples (1:1 ratio), …

BSG Advantage: Consumable ● Not derived from immuno-affinity

Vialaret, Jerome & Kadi, Sarah & Tiers, Laurent & O Flynn, Robin & Lehmann, Sylvain & Hirtz, Christophe. (2018). Albumin depletion of human serum to improve quantitative clinical proteomics. Current Topics in Peptide & Protein Research 19. 53-62. http://www.researchtrends.net/tia/abstract.asp?in=0&vn=19&tid=26&aid=6192&pub=2018&type=3

The article states “In comparison, methods using antibodies needed at least one-half day more. The albumin depletion method allowed to save precious time. ”. The authors concluded that the AlbuVoid™depletion method proved to be faster and more cost-effective than antibody based methods, and could be helpful for biomarker enrichment and detection in medical research.

BSG Advantage: Enrichment/Depletion ● On-Bead Digestion

Liu, Wenli, et al. "Erythroid lineage Jak2 V617F expression promotes atherosclerosis through erythrophagocytosis and macrophage ferroptosis." The Journal of Clinical Investigation (2022).

To explore underlying defects promoting oxidative changes in Jak2VF Red Blood Cells (RBC), unbiased proteomics profiling was conducted. The article states “…haemoglobin removal and on-bead digestion, …was based on the protocol in HemoVoid™ kit. … Samples were first digested with LysC in HVWB (from kit) with protease: protein ratio 1:100 overnight at 37 °C and then with trypsin in HVWB (from kit) with protease: protein ratio 1:30.”. The proteomic data showed a prominent increase in EIF2AK1 (also known as heme-regulated inhibitor, HRI) and EIF2A (right), consistent with increased oxidative stress.

BSG Advantage: Enrichment/Depletion ● On-bead Digestion

Klatt, Stephan, et al. "Optimizing red blood cell protein extraction for biomarker quantitation with mass spectrometry." Analytical and Bioanalytical Chemistry (2020): 1-14. The article describes the advantage of HemoVoid^™ in detection of low abundance proteins when comparing their amounts (in percent) between four alternative extraction conditions, stating "... Most peptides, following HemoVoid^™ extraction, showed ion abundances ranging between 1.00E+5 and 1.00E+6 (31%). In comparison to this, fewer peptides (10–23%) were within this range following extraction with all other protocols". With respect to potential biomarkers for Parkinson’s Disease, the article states "For example, PRDX6 accounts for 0.4% of the total ion abundance after DOC (deoxycholate) extraction, whereas following HV (HemoVoid^™) extraction, this increases to 8%, a 20-fold enrichment". The article describes methods to digest the HemoVoid^™ bead-bound proteome, greatly simplifying the workflow for LC-MS/MS analysis.

BSG Advantage: Functional Integrity

David L. Wang, Chuanguang Xiao, Guofeng Fu, Xing Wang and Liang Li. “Identification of potential serum biomarkers for breast cancer using a functional proteomics technology”. Biomarker Research (2017) 5:11.

The article states “The most dramatic difference for enzyme activity detection in using the AlbuVoid™ for serum protein enrichment was demonstrated in the case of protease activity analysis. Compared with the direct serum proteinase measurement, both the levels and species of proteases were increased significantly in the enriched serum sample. …, and it is necessary to use AlbuVoid™ to enrich these low level proteases to bring them to a high enough level to be detected.”

BSG Advantage: Enrichment / Depletion

Lange, Philipp F., et al. "Annotating N termini for the human proteome project: N termini and Nα-acetylation status differentiate stable cleaved protein species from degradation remnants in the human erythrocyte proteome." Journal of proteome research 13.4 (2014): 2028-2044.

The article describes a goal of the Chromosome-centric Human Proteome Project to identify all human protein species. With 3,844 proteins annotated as “missing” this is challenging. Enucleated and largely void of internal membranes and organelles, erythrocytes are simple yet proteomically challenging cells due to the high hemoglobin content (about 97% by mass) and wide dynamic range of protein concentrations that impedes protein identification. Using a N-terminomics procedure called TAILS, the authors identified 1369 human erythrocyte natural and neo-N-termini and 1234 proteins. From the HemoVoid™ treated, hemoglobin-depleted soluble fraction, 778 proteins were identified, 171 of which were not represented in either the soluble non-depleted fraction or the membrane fraction.

BSG Advantage: Consumable

Chettimada, Sukrutha, et al. "Exosome markers associated with immune activation and oxidative stress in HIV patients on antiretroviral therapy." Scientific Reports 8.1 (2018): 7227. The authors investigated plasma exosomes in HIV patients and their relationship to immunological and oxidative stress. For LC-MS/MS proteomics, the article states “To obtain purified exosome fractions for proteomic analysis,… albumin was depleted with 2 rounds of albumin depletion using AlbuSorb™”. Using untargeted proteomic analysis, Exosomal Notch4 was increased in HIV-positive subjects versus controls and correlated with immune activation markers.

BSG Advantage: Consumable ● Species Agnostic

Nguyen, Anthony T., et al. "UBE2O remodels the proteome during terminal erythroid differentiation." Science 357.6350 (2017). The mechanisms that drive reticulocyte transition to red blood cell in terminally differentiating cells remain largely unclear. During reticulocyte maturation, the proteome is remodeled through the programmed elimination of most generic constituents of the cell, in parallel with abundant synthesis of hemoglobin. The study used multiplexed quantitative proteomics to identify candidate substrates of UBE2O, an E2 (ubiquitin-conjugating) enzyme, in an unbiased and global manner. Because of the overly abundant presence of Hemoglobin, selective depletion of Hemoglobin was necessary. The article states “Reticulocytes were lysed by vortexing for 5 minutes at room temperature… An additional 10 bed vol of Hemoglobind™ suspension was added to the samples, …were … processed for TMT quantification.”

BSG Advantage: Functional Integrity

Cortese-Krott, Miriam M., et al. "Identification of a soluble guanylate cyclase in RBCs: preserved activity in patients with coronary artery disease." Redox Biology (2017). http://www.sciencedirect.com/science/article/pii/S2213231717306535

In brief, the authors aimed to investigate whether RBCs carry a functional soluble guanylate cyclase (sGC) signalling pathway and to address whether this pathway is compromised in coronary artery disease. The article states “Using a commercial resin (HemoVoid™), which removes hemoglobin… and allows enrichment of soluble cytoplasmic proteins, we established a procedure that allows fast and reliable preparation of hemoglobin-free cell lysates from as little as 1-2 ml blood. In those samples, expression and activity of the cGMP-generating sGC, cGMP-hydrolyzing PDE5 and cGMP-transducing PKG was assessed by enzymatic assays and Western blot analysis”

BSG Advantage: Consumable

Taylor SW, Clarke NJ, Chen Z, McPhaul MJ. A high-throughput mass spectrometry assay to simultaneously measure intact insulin and C-peptide. Clin Chim Acta. 2016 Apr 1;455:202-8. doi: 10.1016/j.cca.2016.01.019. The authors simultaneously measured intact endogenous-insulin and derived C-peptide, to help predict development of diabetes mellitus, as well as in differential diagnosis in cases of hypoglycemia. Cleanascite™ is shown both to improve LC-MS measurements, and validated in accordance with CLIA '88 guidelines.

BSG Advantage: Enrichment/Depletion ● Species Agnostic

Jenull, Sabrina, et al. "The histone chaperone HIR maintains chromatin states to control nitrogen assimilation and fungal virulence." Cell Reports 36.3 (2021): 109406. https://www.sciencedirect.com/science/article/pii/S2211124721008196#mmc1. The authors report a pivotal role for the HIR histone chaperone complex in modulating virulence of the human fungal pathogen Candida albicans. The article states for “Cell-free supernatants from 16 hours YNB-BSA (0.025% BSA) cultures grown at 30°C were used for Mass-Spec analysis. Collected supernatants were lyophilized and dissolved in 400 μl of water for AlbuVoid™ treatment for albumin depletion…Albumin-free enriched secretory proteome was eluted from beads”.

BSG Advantage: Functional Integrity

Wang, Xueyu, et al. "Epigenetic Silencing of miR-33b Promotes Peritoneal Metastases of Ovarian Cancer by Modulating the TAK1/FASN/CPT1A/NF-κB Axis." Cancers 13.19 (2021): 4795. To examine whether fatty acids in OCM are the main source of energy for tumors, all fatty acids in OCM were first removed by Cleanascite™ Lipid Removal Reagent. XTT cell viability analysis was performed and showed that the cell growth rate of ES-2 and MES-OV cells was remarkably reduced when cocultured in lipid-depleted OCM and “both miR-33b overexpression and depletion of fatty acids by Cleanascite in OCM significantly impaired ovarian cancer cell migration and invasion.”

BSG Advantage: Enrichment/Depletion

Jing, Lun, et al. "PROTEOMIC ANALYSIS IDENTIFIED LBP AND CD14 AS KEY PROTEINS IN BLOOD/BIPHASIC CALCIUM PHOSPHATE MICROPARTICLE INTERACTIONS." Acta Biomaterialia (2021). Here, in a LC-MS/MS proteomic study, the article describes use of HemoVoid™ and AlbuVoid™ prior to LC-MS analysis, “…After albumin depletion, analysis of the significant deregulated proteins showed that 27 signaling pathways significantly changed in blood cells…”

BSG Advantage: Enrichment/Depletion

A Preliminary Investigation Using Targeted LC-MS Proteomic Methods Demonstrates Unique Serum Profiles of Hospitalized SARS-CoV-2 Patients https://www.biotechsupportgroup.com/v/vspfiles/templates/257/pdf/ASMSBSGLFPoster.pdf

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, AlbuSorb™ PLUS depletion methods were used to characterize the functionality of the innate immune response in hospitalized Covid-19 patients, more precisely than current methods.

BSG Advantage: Consumable

Zheng, Wenshu, et al. "Nanopore-based disease diagnosis using pathogen-derived tryptic peptides from serum." Nano Today 45 (2022): 101515. An immunoprecipitation-coupled nanopore (IP-NP) analysis of samples resulted in frequent and prolonged pore blockades and unstable current baselines. This appeared to result from lipid or lipoprotein complexes that were non-specifically adsorbed by the immunoprecipitation matrix. This was markedly reduced when samples were lipid extracted before immunoprecipitation. The article states “Treatment of digested serum samples with two commercial lipid removal agents revealed variable effectiveness in reducing these artifacts, with one reagent (LRA; synthetic calcium silicate hydrate) revealing little to no effect …Serum digests treated with the second reagent (Cleanascite™) demonstrated the absence of these artifacts”. The article states “FP-10pep αHL signals detected from serum CFP10pep immunoprecipitates following effective lipid-depletion revealed blockade current and dwell time characteristics similar to those observed … from PBSsuspensions, and were not influenced by several factors responsible for clinical interferences.”

BSG Advantage: On-Bead Digestion ● Species Agnostic

Zheng H, Zhao C, Qian M, Roy S, Arpa A, et al. (2015) AlbuVoid™ Coupled to On-Bead Digestion – Tackling the Challenges of Serum Proteomics. J Proteomics Bioinform 8: 225-230. The article describes new methods using a proteome-enriching product, AlbuVoid™, which depletes Albumin through a negative selection or voidance strategy, retaining the vast amount of the remaining serum proteome on the bead. Then the enrichment is combined with a direct and seamless integration with Trypsin digestion, a method conventionally referred to as on-bead digestion. The digest time was evaluated as a parameter to identify whether different sub-populations of peptides and proteins can be observed by LC-MS analyses. Using 2 different 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 workflow efficiencies over other high abundance depletion and in-solution digestion workflows.

BSG Advantage: Functional Integrity ● Species Agnostic

Wilhelm, Christina M., et al. "A comprehensive evaluation of the efficacy of leading oxime therapies in guinea pigs exposed to organophosphorus chemical warfare agents or pesticides." Toxicology and applied pharmacology 281.3 (2014): 254-265. Acetylcholine is an essential neurotransmitter, and inhibitors of cholinesterases(ChEs) are potent toxins. The objective of the present study is to identify an oxime antidote, under standardized and comparable conditions, that offers protection against chemical warfare agents or pesticides. Clinical signs of toxicity were observed for 24 h post challenge, and blood cholinesterase was measured by a modified Ellman’s enzymatic assay. To remove colorimetric interference in the assay, the article states “Terminal blood samples were collected and processed for all survivors using HemogloBind™”.

BSG Advantage: Functional Integrity ● Species Agnostic

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). The authors aimed at elucidating the components of multiprotein complexes on a proteome-wide scale, based on biochemical fractionation of native soluble macromolecular complexes followed by tandem mass spectrometry to identify components. The article states “…clarified heart and liver homogenateswere …, depleted of hemoglobin using HemogloBind™”. A second Biotech Support Group product, formerly called SeraFILE PROspector and now trademarked as NRicher™ 6, was used for sub-proteome enrichment. The article states “…bead-based sample pre-separations were performed for C. elegans lysates as per manufacturer’s instructions…fractions (NRicher™ 6 eluates) were subjected to ion exchange fractionation…”. The authors conclude that many complexes are conserved across species, and by correlating the results with genome sequence information, are able to predict more than one million interactions in 122 eukaryotes.

Biotech Support Group (BSG) is a leading manufacturer and supplier of sample preparation products.