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Rapid, Semi-automated Immunopeptidome profiling

Rapid, Semi-automated Immunopeptidome profiling

In 2022 MS Wil has also become supplier of ReSyn Biosciences’ MagReSyn microparticles. These microparticles are build up on a completely new microparticle technology platform. ReSyn’s new technology comprises a hyper-porous polymer matrix that allows penetration of biological and synthetic molecules throughout the volume of the microparticles. This offers exceptionally high surface area for binding of molecules and allows performance that is orders of magnitude greater than alternate technologies. MagReSyn microparticles are therefore very suitable for the development of new methods for achieving high biomolecule binding capacity (enabling miniaturization and high-throughput processes), for different kinds of applications.

In that light, Monash University in Melbourne developed a rapid, semi-automated immunopeptidome profiling method for the Major Histocompatibility Complex class I and II (MHC-I and MHC-II) associated peptides. In this article we will provide a short summary of their research and results.

Introduction

In collaboration with ReSyn Biosciences, the Monash University in Melbourne, Australia developed a rapid, semi-automated immunopeptidome profiling method for the Major Histocompatibility Complex class I and II (MHC-I and MHC-II) associated peptides.

The MHC system is also known as the human leukocyte antigen (HLA) in humans. HLA bound peptides play a crucial role in the development of adaptive immune response in vertebrates. Unlike the innate immune system, which reacts to a broad range of non-specific pathogens. The adaptive immune system reacts highly antigen specific. Because these immunopeptides play a key factor to the specific immune response, its understanding has the potential for a wide variety of developments like precision/personalized immunotherapies for cancer, autoimmune diseases and infections.

For immunopeptidome profiling, there are however several analytical challenges, such as peptide isolation uncertainty, suboptimal identification rates, lack of experimental and computational standards, and lack of accessibility to large-scale community-generated datasets. But with the help of MagReSyn® Protein A in the described method, these challenges could be tackled including working with low input material, increasing throughput and reproducibility.

MagReSyn® is a newly developed novel (patented) magnetic microparticle technology platform, comprising a hyper-porous polymer matrix that allows penetration of biological and synthetic molecules throughout the volume of the microparticles (as shown in figure 1). This offers an exceptionally high surface area for the binding of molecules and allows performance that is orders of magnitude greater than alternate technologies, that use porous or cracked microparticles where the binding of biological molecules is limited to the surface. The binding capacity serves as a major performance contributor to the vast array of techniques in the life sciences like bioseparation, diagnostics, DNA sequencing, flow cytometry, drug discovery, proteomics, and genomics.

Figure 1. Different microparticle technologies used in mass spectrometry sample preparation.

Methods

In this method, MagReSyn® Protein A MAX is used as microparticle. This microparticle contains the covalently coupled surface protein, Protein A, for highly specific purification of immunoglobulin G (IgG) antibodies and for immunoprecipitation experiments. Protein A MAX is specially engineered for custom applications where maximum antibody binding is desired.

This described semi-automated method for MHC molecule immunoprecipitation covers all the steps from anti-MHC antibody cocktail loading on the MagReSyn® Protein A MAX magnetic microparticles, to the elution of purified MHC Class I and II complexes. Furthermore, this method can process up to 12 samples in parallel but can be scaled up to 96 samples without the requirement for method re-optimization. The image below schematically describes this semi-automated method (see figure 2).

Figure 2. Semi-automated workflow for MHC molecule immunoprecipitation

Results

This method shows high throughput for immunopeptide enrichment where the automated workflow has a total run time of approximately 120 min that allows for:

  1. Loading anti-HLA antibodies on MagReSyn® Protein A MAX microparticles.
  2. Removal of non-specifically captured anti-HLA antibodies.
  3. Enrichment of HLA Class I and II peptides form a cell lysate and their elution.

Furthermore, high reproducibility and enrichment efficiency were achieved whilst processing 12 samples in parallel on KingFisher™ Duo magnetic handling station. This throughput can be further scaled up to 96 samples in parallel and processed in approximately 120 min, by utilising a KingFisher™ Flex or Apex magnetic bead handling stations, without the need for additional method re-optimization.




Part Numbers & Product Specifications:

SupplierProduct Nr.Specifications
ReSyn BiosciencesMR-PAM002MagReSyn Protein A MAX – 2ml custom product
MR-PAM005MagReSyn Protein A MAX – 5ml custom product
MR-PAM010MagReSyn Protein A MAX – 10ml custom product




Authors & Other Sources:

Terry Lim & Pouya Faridi
Translational Antigen Discovery Lab, and Monash Proteomics and Metabolomics Facility, Monash University, Melbourne, Australia.

Stoyan Stoychev & Justin Jordaan
ReSyn Biosciences (Pty) Ltd.

More info about ReSyn’s microparticle technology: https://resynbio.com/resyntechnology/
More info about Protein A MAX: https://resynbio.com/protein-a/

Full research article: ImmunoPep-App-note-16-Sep-2022-A5.pdf (mswil.com)

We, at MS Wil, supply a wide range of magnetic microparticles from ReSyn Biosciences, including Protein A MAX, HILIC, SAX, Streptavidin, Trypsin, and many more. Click here to see our full offer of magnetic microparticles in our online catalog.


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