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Designing a Proteomic Biomarker Study: Approaches for Exploration and Follow-up

This blog post was written in partnership with Ray Biotech, who provides cutting-edge array technologies that accelerate the discovery of disease-related protein biomarkers and key factors, the identification of new drug targets and the advancement of personalized medicine. In this blog post, Ray Biotech provides an in-depth explanation of common research questions regarding proteomic biomarker studies. Their services are available on the marketplace.

Proteomics is the study of the interactions, function, composition and structures of proteins. It provides a better understanding of current cellular activity than genomics. The goals of a proteomic study may be to understand a disease mechanism, evaluate molecular responses to therapies, establish diagnostic criteria, assess cellular functions and more.

In choosing an approach to answering your research questions, it’s important to choose the best analytical methodology for your biospecimens and goals. In this blog, we walk through the most important considerations when planning your biomarker study with affinity proteomic methods.

What protein analysis methodologies do you offer?

Expression Profiling

RayBiotech is a leading manufacturer of antibody arrays and other affinity proteomic platforms. We have developed platforms at several levels of size, scope and sensitivity to suit a wide range of budgets and objectives. The first step for most protein biomarker studies is expression profiling for identification of differentially expressed proteins in a disease vs. a control population. This can be done at the broadest scale using our antibody array panels spanning 1000-8000 proteins. If quantitative output is desired, the researcher may choose a 1,200-target human panel, a 640-target murine panel or a 282-target rat panel all of which employ sandwich antibody pair immunodetection to achieve highest sensitivity and specificity. To achieve highest content, semi-quantitative panels are available: 8,000 human proteins, 2,808 murine proteins, 1,500 rat proteins or 500 rabbit proteins; these platforms use solid-phase immunocapture with direct biotin-labeling.

The researcher may also choose to profile a smaller panel of targets (10-200 proteins) focusing on changes in certain biological pathways, processes or molecule types. RayBiotech manufactures a wide variety of pre-validated array kits with panels curated by research area. Thus, the choice of array panel should reflect 1) the molecules of interest, 2) how expansive or targeted the researcher needs the initial query to be and 3) whether quantitative output is essential for the study. For example, Dorien et al. utilized the Human L1000 Array (a 1000-protein semi-quantitative screening array), along with the Quantibody® Human Cytokine Array 640 (a 640 protein quantitative array) to identify possible biomarkers in plasma samples from endometriosis patients. The array identified 4 differentially expressed proteins that were then validated by quantitative ELISA, and 1 protein was identified as a potential biomarker (Reprod Sci. 2020 Feb;27(2):751-762).

Representative images depicting analysis of 2 mouse serum samples with the RayBio Mouse L-Series Array [AAM-BLG-2], which detects 500 proteins.

PTM Profiling

Besides overall expression, the various proteoforms of a biomarker may be relevant in the disease process. This can be interrogated by another discovery approach: proteoform profiling, which detects post-translational modifications on a large scale. For this purpose, RayBiotech’s unique range of post-translational modification (PTM) arrays use high density antibody array panels (500-8000 proteins) with specific labeling chemistries or affinity agents to identify the nitrosylation, acetylation, palmitoylation, oxidation, glycosylation or phosphorylation statuses of each protein in the panel. Proteoform identification is the first step toward understanding the functions of the specific proteoforms as well as discovering molecular signatures for disease diagnosis or identifying possible drug targets.

Following Up

After exploratory studies have identified a single or small subset of candidate proteins, tracking changes over time or across experimental groups may be needed. For this purpose, RayBiotech also provides single-plex assays for precise, sensitive protein analysis. Options include quantitative ELISA kits or Western blot service, both of which can detect both protein expression and phosphorylation or other PTMs. For biomarker candidates that are low-abundance and difficult to detect, two ultrasensitive methods are available. RayBiotech’s Immuno-PCR ELISA (IQELISA®) is a standard sandwich-based immunoassay that employs a DNA oligo to label the immunocomplex, allowing a real time PCR read-out. This format typically yields 10-fold lower LOD compared with the corresponding colorimetric ELISA. If further sensitivity is needed, RayBiotech offers full service single-molecule protein assay (SIMOA), which can detect down to the femtogram (fg/mL) level. This assay uses antibody-coated beads, a fluorescent-conjugated detection antibody, and digital readout to achieve quantification of biomarkers at concentrations that are difficult or impossible to measure by any other method.

How do I determine the best approach for my sample type and research goals?

The determination of optimal approach can depend on several factors related to the inputs, including the sample number, type and volume available for the study. The outputs, such as group comparisons, questions and goals for the study should also be considered. However, because the amount of sample available dictates which approaches are possible, the sample volume should be determined first as a matter of practicality.

Sample Volume

The amount of sample required for analysis depends on the sample dilution and type of assay used. In affinity proteomics, a phenomenon called “sample matrix effect” (SME) can occur, which affects the ability of an antibody to bind to its target molecule, confounding the results. Therefore, we recommend at least 2-fold fold dilution for any biological sample. For quantitative proteomic expression profiling of 1200 proteins, 1.5 mL of serum/plasma and other biological fluids or 3 mL of cell culture media is needed. Alternatively, semi-quantitative proteomic expression profiling of 8000 proteins requires only 40 µl serum/plasma and other biological fluids or cell culture media. For cell or tissue lysates, good starting points are: 500 µL of lysis buffer per 1 million cells or 10 mg tissue; for cell culture media samples, seed approximately 1 million cells in a 100 mm tissue culture plate, then culture for 5 days. Therefore, the starting material and volume can dictate the downstream approach.

Sample Type

Whether the assay is validated or suitable for use in the sample type available is also an important consideration when determining the best approach. RayBiotech platforms can measure protein across 15 different species, including many common mammalian and aquatic species (human, mouse, rat, non-human primate, cow, horse, dog, cat, sheep, chicken, pig, rabbit, zebrafish, dolphin) as well as bacterial and viral strains (E. coli, host cells, SARS-COV-2, MPOX, HPV, HCV, HIV, etc.).

RayBiotech validates protein analysis methodologies in pooled, normal serum and plasma samples, and depending on the particular assay, has also tested them in a variety of sample types including cell culture media, cell and tissue lysates, urine, tears, saliva and other biological fluids. Publications citing RayBio® arrays used with alternate sample types can be found in our publication database, which may provide further guidance on the suitability of the array for the particular sample type. For example, Jovanovic et al. utilized the RayBio L6000 Array with osteoblast lysates from control and type XIV osteogenesis imperfecta patients. This approach identified differentially expressed proteins and revealed that proteins related to cell adhesion were affected, which complemented transcriptomic data (Matrix Biol. 2023 Aug:121:127-148.). In another study, Weeding et al. used the RayBio Kiloplex (a 1000-target quantitative array) for proteomic analysis of urine samples from lupus nephritis patients. Here, the array identified a urinary signature that was associated with specific treatments, which may inform drug monitoring strategies in the future (Lupus Sci Med. 2022; 9(1): e000763).

Growth factor expression in vitreous humor samples measured by the Quantibody Human Growth Factor Array.

How To Choose

In addition to the factors noted above, the design principle/format of the array platform can also be considered when choosing the best approach. Antibody arrays can be cytometric bead-based, (read by flow cytometry), nitrocellulose membrane-based (read by chemiluminescent imaging) or glass slide-based (read by laser microarray scanner). Thus, the researcher’s available detection equipment may dictate which array type is most practical to use, should they decide to run the array in their own lab.

RayBiotech’s How to Choose an Array infographic includes a visual summary of these factors to help guide your decision. The infographic can be downloaded here. For an in-depth technical comparison of the various array platforms, please refer to this article.

How do I analyze/interpret my data?

Once you have established your approach, determined the experimental design and completed the experiment, determining how to analyze and interpret large, complex datasets encompassing hundreds of samples and thousands of proteins can be difficult. Many hours can be lost exploring the data without successfully answering the important research questions the study set out to ask. RayBiotech’s standard analysis report contains raw and processed data, including final concentration or signal intensity for each analyte and each sample. These data are already cleaned and standardized, allowing for a seamless transition into deeper statistical analyses if needed.

Biostatistical Services

You also have the option of consulting with RayBiotech’s bioinformatics experts to build a custom analysis report for your proteomic or genomic data. Whether you need cluster analysis, pathway analysis or biomarker selection, we can help find the hidden answers to your research questions. Our capabilities also include integration of multiple data sets (NGS, RNA Seq, metabolomic profiles, etc.) using a variety of methods, including predictive modeling, mediation, GO/KEGG association, network analysis and more.

As the first company to commercialize the protein array and an innovative leader in multiplex protein analysis, RayBiotech has both the right tools and expertise to successfully advance your biomarker discovery projects. RayBiotech’s team of scientists and technical consultants can guide you through experimental design, choosing the best assays, conducting the experiment and data analysis. If you have an idea for a project, send us a request through the portal.