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GELFrEE 8100® Fractionation System in a High-Throughput Pipeline for Validation of Antibodies

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Introduction

Western blot is a widely used method of analysis to establish antibody specificity. However, the Western blot protocol is inherently restrictive as it is low in both throughput and accuracy, generally difficult to standardize (SDS PAGE followed by transfer / immunoblotting), and usually applies to the analysis of one antibody at a time. An image of a Western blot analysis / immunoblot is habitually shown alongside a commercially available antibody to document its specificity, but it does not represent definitive evidence as to quality, which is often unpredictable. Off-target binding and cross reactivity is evident in around 50% of all commercial antibodies, making them unsuitable or redundant for their intended application. Such a lack of specificity fuels a costly waste in both time and resources through unproductive experiments and concerns regarding false outcomes.

The authors of this study were members of an international working group on antibody validation (IWGAV). This IWGAV proposed a five pillar concept that might provide a more stringent quality assessment. They sought to develop a ‘capture’ format for Western blot analysis (PAGE-MAP), using a combination of preparative gel electrophoresis (PAGE) and microsphere affinity proteomics (MAP). Expedeon’s GELFrEE 8100 Fractionation System was used to collect soluble, biotinylated and size separated protein fractions to enable parallel readouts with antibody arrays, shotgun mass spectrometry (MS), and immunoprecipitation (IP) followed by MS (IP-MS), to satisfy some of the pillars suggested. These pillars included:

1. Targeted disruption of DNA or RNA to generate negative controls
2. The use of MS data as a reference
3. The use of different antibodies to the same protein as references for each other
4. Expression of tagged proteins to generate positive controls
5. IP-MS.

The outcomes of this study demonstrated that traditional methods for antibody validation (preparative SDS PAGE followed by Western blotting), could be converted to a high-throughput ‘capture’ format (i.e. PAGE-MAP). PAGE-MAP offers higher precision for comparing antibody reactivity profiles, and enables direct assessment of antibody specificity via PAGE–IP-MS.

Results

In this study, the authors sought to convert the traditional Western blot protocol to a ‘capture’ format for high-throughput antibody validation, to potentially overcome the inherent and restrictive limitations associated with this technique.

Six cell lines (human and primary), were harvested and lysed, and the resulting protein was biotinylated (300µg protein for each cell line). Following desalting, sample proteins were subjected to preparative PAGE followed by size separation, with the GELFrEE 8100 Fractionation System, resulting in 12 soluble fractions, i.e. 72 in total across six cell lines.

PAGE-MAP (array-based Western blot)

A 50µl aliquot from each fraction was incubated with bead-based barcoded antibody arrays for multiplexed IP. Captured (immobilized) proteins were labeled with streptavidin conjugated to a fluorescent reporter, R-phycoerythrin (RPE), for ‘on-bead’ detection by flow cytometry. Fluorescence reactivity peaks obtained across the biotinylated and size separated protein fractions were shown to correlate with antibody targets in the subsequent analysis. Thus, in a similar manner to bands obtained in Western blot analysis, the reactivity peaks from PAGE-MAP represent antibody reactivity. The protein fractions used for PAGE-MAP were also compatible for further parallel assessment with both shotgun MS and IP-MS.

Shotgun MS (PAGE-MAP–MS)

Following PAGE-MAP, a second aliquot from each fraction was subjected to ‘on-bead’ trypsin digestion to generate peptides for PAGE-MS (PAGE-MAP–MS). Subsequent analysis yielded size distribution profiles for thousands of proteins as peaks across the fractions and was shown to have a positive predictive value of 88–90% for both Western blot and PAGE-MAP analysis.

IP-MS (PAGE–IP-MS)

The results obtained from both PAGE-MAP and PAGE-MS, i.e. signal-to-noise ratios and correlations between antibody reactivity patterns and MS data, were assessed and selected antibodies used in single-plex IP from enriched fractions to definitively assess specificity and directly identify antibody targets by IP-MS. (As a control, and to monitor single-plex IP, an aliquot of the beads was labeled with streptavidin-RPE for detection of biotinylated bound protein by flow cytometry). The results demonstrated a clear association between streptavidin-RPE fluorescence intensity following IP and the successful identification of antibody targets by MS. Furthermore, the amount of intended target, as measured by PAGE-MS, was predictive of successful target identification by single-plex IP-MS.

Together, the results demonstrate that PAGE-MAP is a powerful method to identify antibodies that react with the same protein and as a suitable alternative to Western blot analysis.

Summary and Conclusion

In this study, which arose due to the unpredictable quality of commercially available antibodies, the authors (members of an IWGAV) proposed five different ‘conceptual pillars’ to make up a more rigorous quality assessment for commercially available antibodies compared with analysis using more traditional Western blot methods.

Using GELFrEE® 8100 Fractionation System to collect soluble, biotinylated and size separated protein fractions, they were able to develop a high-throughput pipeline, PAGE-MAP, for the validation of antibodies and those that bind the same denatured protein.

They also demonstrated the potential applicability of PAGE-MS data to be used as a reference to obtain a rough estimate of specificity by PAGE-MAP–MS correlation analysis, and as a predictive guide to successful target identification by single-plex IP-MS.

GELFrEE® 8100 Fractionation System

In this study the authors relied on the GELFrEE 8100 Fractionation System to collect soluble, biotinylated and size separated protein fractions. The fractions facilitated the development of a parallel readout with antibody arrays, shotgun MS, and IP-MS.

The GELFrEE® 8100 Fractionation System offers several advantages:

• Intact protein MW fractionation, isolation, and purification
• Liquid phase recovery without band or spot cutting
• Broad mass range – Fractionation up to 500kDa
• Up to eight samples processed in parallel
• Programmable fractionation for isolating and purifying targeted proteins
• High protein recovery (>80%)
• High reproducibility (>15% CV)
• Sampling unbiased by hydrophobicity, pI
• High loading capacity (>5X more than a 1D gel)
• Proteins are recovered intact, for complete characterization.

Applications

• Simplify and reduce the dynamic range of complex protein mixtures for bottom up discovery proteomics using LC-MS / MS
• Fractionate and recover proteins intact for top down proteomics
• Isolate and enrich user-selected molecular weight fractions for targeted protein quantification using LC-MS / MS
• Isolate intact proteins to analyze PTMs, variants and alterations
• Separate protein pull down components for target protein purification
• Separation, isolation, and recovery of intact antibodies for in depth characterization.

All the above are key for enabling effective programmable fractionation, isolation and purification across a broad mass range.

Reference

Khairalla, AS., Omer, SA., Mahdavi, J., Aslam, A., Dufailu, OA., Self, T., Jonsson, AB., Geörg, M., Sjölinder, H., Royer, PJ., Martinez-Pomares, L., Ghaemmaghami, AM., Wooldridge, KG., Oldfield, NJ., Ala’Aldeen, DA. Nuclear trafficking, histone cleavage and induction of apoptosis by the meningococcal App and MspA autotransporters. Cell Microbiol. 2015 Jul;17(7):1008–20. doi: 10.1111/cmi.12417. Epub 2015 Feb 8. Available at: https://www.ncbi.nlm.nih.gov/pubmed/?term=Nuclear+trafficking%2C+histone+cleavage+and+induction+of+apoptosis+by+the+meningococcal+App+and+MspA+autotransporters Accessed November 2018.