Proteomics experiments have typically high economic and technical barriers to broad biomedical scientists, which not only result in costly supplies and accessories for sample preparation but also the reluctance to adapt new techniques. In the present study, we present an effective and efficient, yet economical technology, which we call E3technology, for proteomics sample preparation. By immobilizing silica microparticles into a polytetrafluoroethylene (PTFE) matrix, we developed a novel medium, which could be used as a robust and reliable proteomics platform to generate LCMS-friendly samples in a rapid and low-cost fashion. Using different formats of E3technology, including E3tip, E3filter, E3cartridge, and E3plate, we explored a variety of sample types in varied complexity, quantity, volume, and size, including bacterial, fungi, mammalian cells, mouse tissue, and human body fluids. We benchmark their performance against several established approaches. Our data suggest that E3technology outperforms many of the currently available techniques in terms of proteome identification and quantitation. It is widely applicable, highly reproducible, readily scalable and automatable, and is user-friendly and stress-free to non-expert proteomics laboratories. It does not require specialized expertise and equipment, and significantly lowers the technical and economical barrier to proteomics experiments. An enhanced version, E4technology, also opens new avenues to sample preparation for low input and/or low-cell proteomics analysis. The presented technologies by our study represent a breakthrough innovation in biomedical science, and we anticipate widespread adoption by the proteomics community.

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Nucleotide‐binding oligomerization domain‐containing protein 2 (NOD2) is an intracellular innate immune receptor expressed in epithelial cells and macrophages. NOD2 is activated and stabilized upon binding the peptidoglycan fragment muramyl dipeptide (MDP). The binding process initiates an innate immune response leading to the release of proinflammatory molecules via the activation of transcription factors including NF‐kB pathway. Three Single‐nucleotide polymorphisms of NOD2 are correlated with an increased susceptibility to Crohn's disease, one of the known forms of Inflammatory bowel diseases (IBD). NOD2 CD variants are inherently unstable; however, stability can be rescued by overexpressing NOD2 interacting partner, HSP70 and through thiamet G induced increase of global O‐GlcNAc level. This study is focused on identifying O‐GlcNAc sites of NOD2, revealing other binding partners, and understanding how these processes, guide NOD2 regulation. In working towards this goal, mass spectrometry and co‐immunoprecipitation experiments were performed to identify O‐GlcNAc modification site and a novel interacting partner of NOD2 respectively. Initial data obtained demonstrated that OGT is a novel binding partner of NOD2, in complex with HSP70 and guides OGT to the NOD2 substrate. Interestingly, the data suggest the complex is changed upon NOD2 engagement with peptidoglycan fragment, MDP. This highlights that there are multiple control mechanisms involved in NOD2 regulation. Future work will investigate the position of OGT and HSP70 binding on NOD2.

The innate immune system is the body's first line of defense against invading pathogens such as bacteria. Innate immune receptors such as nucleotide‐binding oligomerization domain‐containing protein 2 (NOD2) are crucial in recognizing conserved patterns derived from bacterial cell walls as bacteria break down, which induces immune responses via signaling pathways such as nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB). Variations in NOD2 have been linked to Crohn's disease (CD), and our recent biophysical and biochemical studies have revealed that these variants are inherently unstable, and hence, they might be subject to unwanted degradation in cells. This leads to insufficient “sensors” available for recognizing bacterial invasion followed by uncontrollable inflammation in CD. Interestingly, these negative outcomes could be lessened or even reversed by overexpression of chaperone heat shock protein 70 (HSP70), a novel regulator of NOD2, to enhance the half‐lives of NOD2 variants and enable their activation of immune signaling pathways. The ability of HSP70 to “correct” NOD2 variants’ function indicates this chaperone has the potential to become a therapeutic drug against CD. Therefore, we hypothesize that elucidating the mechanism of HSP70‐NOD2 interaction will provide useful information for developing a peptide‐based drug that can stabilize NOD2 variants in CD patients and rescue their function. We have designed several constructs serving as small peptide analogs of HSP70 for characterizing the HSP70‐NOD2 interactions. From our trypsin‐mediated degradation assay, we show that these analogs retain HSP70's ability to stabilize and protect NOD2 against trypsin digestion. We then measure the binding affinities of NOD2‐HSP70 constructs interactions in the absence or in the presence of different cofactors (ATP, ADP, NOD2 ligand MDP) using surface plasmon resonance (SPR). In the future, we plan to determine the mechanistic details of NOD2‐HSP70 interaction utilizing cross‐linking/mass spectroscopy experiment. The findings from this study will enhance our understanding of NOD2 regulation and open up opportunities for expanding the development of effective therapeutic drugs against CD.