Autoimmune diseases affect >20 million people in the US today. Currently, disease‐specific autoantibodies are thought to be the best biomarkers for diagnosis. Conventional immunoprecipitation methods have been used to identify autoantigens from the most common autoimmune diseases. However, these diseases account for only 6.5 million of the 20 million patients suffering from autoimmune diseases, leaving many without diagnoses until irreversible damage occurs. The remaining 13.5 million patients have >70 autoimmune disorders without well characterized autoantibodies. The state‐of‐the‐art diagnostic test of these remaining diseases relies on gel electrophoresis of immunoprecipitated radiolabeled proteins, which cannot be identified by MS due to safety issues and the overwhelming presence of immunoglobulins. We have created an immunoprecipitation method that uses serum from patients with any autoimmune disorder to identify patient‐specific autoantigen proteins. This method uses a reversible click chemistry tag, called ProMTag. One end of the ProMTag forms a reversible, covalent bond with protein by coupling to lysines and amino termini. The other end of the ProMTag can form an irreversible, covalent bond with a solid bead support via a click chemistry, methyltetrazine‐TCO, pairing. In this study, the proteins of cell lysates that contain potential autoantigens were labeled with ProMTag. The ProMTagged‐proteins were exposed to patient antibodies bound to Protein A beads, thus capturing the ProMTagged autoantigens. All proteins were released from the Protein A beads, including ProMTagged‐autoantigens and untagged‐antibodies. The ProMTagged‐autoantigens were subsequently coupled to TCO beads, and the untagged‐antibodies were washed away. The linkage between the ProMTag and autoantigens was then reversed, yielding autoantigen proteins with greatly reduced antibody contamination ready for MS analysis. MS analysis successfully identified autoantigens from patient serums with rheumatoid arthri. This autoimmune biomarker discovery method can accelerate sample testing for known autoantigens and facilitating rapid discovery of novel autoantigens for both diagnostic and predictive biomarkers.
Extracellular vesicles (EVs) are complex, cell‐derived nanoparticles generated by all cell types. EVs are composed of lipid bilayer membranes and their associated membrane proteins, nucleic acids, and luminal proteins. The mechanism by which Gram‐positive bacteria shed EVs is still unknown. EVs from the Gram‐positive human pathogen S. pneumoniae, which is a major cause of otitis medi and pneumonia, are of particular interest because of how they EVs modulate the host immune response. To uncover possible mechanisms for EV production and shedding in S. pneumoniae, we have performed a comparative proteomics analysis of EV membrane proteins versus whole‐cell membrane proteins. Membrane proteins were enriched from intact S. pneumoniae cells or their EVs using a ProMTag labeling and capture workflow. ProMTag is a bifunctional protein tag where one moiety of the tag is able to form a reversible, covalent link to primary amines on proteins. The other moiety is methyltetrazine, which can form an irreversible, covalent bond with trans‐Cyclooctene (TCO) on the surface of beads to capture ProMTagged proteins for cleanup and elution. Using this workflow plasma membrane proteins can be tagged, captured, washed to remove non‐plasma membrane proteins, and then eluted in their original, unmodified state. In this study, intact cells and EVs from S. pneumoniae cultures were separated and the extracellular domains of membrane proteins in these two fractions were labeled with ProMTag. The membrane proteins were then enriched, washed, and eluted using the ProMTag workflow. These membrane protein populations were then TMT labeled and analyzed using mass spectrometry. Comparative analysis revealed membrane proteins that are concentrated or absent in EV membranes relative to bulk plasma membrane from whole cells, indicating a selective process for EV formation in S. pneumoniae. With this information, we present a new model for EV formation and shedding in S. pneumoniae.
O‐linked glycosylation, which is regulated by the enzyme GalNAc transferase, plays a vital role in cellular function by regulating membrane protein composition and sorting. Changes in O‐linked glycosylation are very common in diseases such as cancers, diabetes, and Alzheimer’s. It is important to study the effects of changes in O‐link glycosylation on cell membrane composition and function to understand how these changes affect the onset, phenotype, and progression of these diseases. However, membrane proteins are notoriously challenging to analyze at the proteomic level due to their hydrophobicity and the inability to separate the membrane proteins from other proteins in the cell. Here we introduce a new sample preparation workflow that enriches membrane proteins for mass spectrometry analysis to understand changes in protein composition of the plasma membrane as the result of changes in O‐linked glycosylation. To do this, we used the bifunctional tag ProMTag to label and capture plasma membrane proteins via their extracellular domains in cells treated with GalNAc Transferase inhibitors. One end of the ProMTag forms a reversible, covalent bond with protein the other end of the ProMTag is capable of forming an irreversible, covalent bond with a solid bead support via the click chemistry pair of methyltetrazine and trans‐Cyclooctene (TCO). The extracellular domains of proteins from intact wild type HEK cells and HEK cells treated with GalNAc Transferase inhibitors were ProMTagged, captured, cleaned up, and eluted for comparison by mass spectrometry (MS) analysis. MS confirmed enrichment of plasma membrane proteins after sample preparation and protein changes in the plasma membrane as a result of GalNAc Transferase inhibition were characterized. This work establishes a method for enrichment of plasma membrane proteins and reveals how changes in O‐linked glycosylation via GalNAc Transferase inhibition leads to changes in plasma membrane protein composition due to altered membrane protein stability and sorting.
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