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Development of Automated Proteomic Workflows Utilizing Silicon-Based Coupling Agents

ABSTRACT: This work highlights an automated method for enzyme immobilization via 4-triethoxysilylbutyraldehyde (TESB) and 11-triethoxysilylundecanal (TESU) derived silicone-based coupling agents. From the various coupling strategies we scrutinized, TESB and 4-triethoxysilylbutanoic acid (TESBA), the carboxylic acid derivative, were determined to be the most effective. The resulting immobilized enzyme particles (IEPs) displayed robustness and rapid digestion, while also implementing a more streamlined IEP washing procedure. The physical size of IEPs did not correlate to digestion efficiency, providing insights for potential cost-saving’s in enzyme utilization. Immobilization efficiency was determined to be 50.8 ± 7.7% as validated by Bradford's assay. Furthermore, we adapted the IEP procedure into an automated format with a Bravo liquid handler. This allowed for multiple enzymes, and/or coupling agents to be fabricated at once on a 96-well microplate, in a fraction of the time. The automated trypsin TESB and TESBA IEPs rivaled the classical in-gel digestion method for the analysis of Jurkat cell lysate, achieving similar protein group identifications. Moreover, pepsin IEPs favored cleavage at leucine (>50%) over phenylalanine, tyrosine, tryptophan, and methionine residues. Further, when miscleavages occurred, they tended to be at tyrosine residues, or when two aromatic residues were adjacent. The IEP method was then adapted for an in-situ immobilized enzyme microreactor (IMER) fabrication. We determined that TESBA could serve two roles by functionalizing the silica capillary's inner wall, and simultaneously acting as an enzyme coupler. The IMER digestion of bovine serum albumin (BSA), mirroring IEP digestion conditions, yielded a 33-40% primary sequence coverage per LC-MS/MS analysis in as little as 15 minutes. Overall, our findings underscore the potential of both IEP and IMER methods, paving the way for automated analysis and a reduction in enzyme waste through reuse, thereby contributing to a more cost-effective and timely study of the proteome.

INSTRUMENT(S): timsTOF SCP

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Jurkat Cell

SUBMITTER: Maor Arad

LAB HEAD: Leonard Foster

PROVIDER: PXD051564 | Pride | 2024-06-10

REPOSITORIES: Pride

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Project description:Gene expression analyses of three-week old soil grown wild-type (Col-3) and apum23-1 mutant. Expression profiling was conducted as an attempt to identify the genes differentially regulated in apum23-1 mutant This study was under taken to characterize a member of pumilio family, APUM23 in Arabidopsis. Pumilio, an RNA-binding protein (RBP) containing tandem repeat PUF domains, has been known to repress translational activity in early embryogenesis and polarized cells of non-plant species. Although Pumilio proteins have been characterized in many eukaryotes, their roles in plants are unknown. We detail the characterization of an Arabidopsis Pumilio gene, APUM23. APUM23 is constitutively expressed with higher level in metabolically active tissues, and upregulated in the presence of either glucose or sucrose. The T-DNA insertion mutants apum23-1 and apum23-2 showed slow growth with serrated and scrunched leaves, abnormal venation pattern, and distorted organization of the palisade parenchyma cells, a phenotype reminiscent of nucleolin and ribosomal protein gene mutants. Intracellular localization studies indicate that APUM23 predominantly localizes to the nucleolus. Based on the localization, rRNA processing was examined in apum23 mutant. In apum23, 35S pre-rRNA and unprocessed 18S and 5.8S poly(A) rRNAs were accumulated without affecting steady-state levels of mature rRNAs, indicating that APUM23 participates in the degradation of rRNA by-products. The apum23 mutant showed increased levels of 18S rRNA biogenesis-related U3 and U14 snoRNAs, and accumulated RNAs within nucleolus. Our results suggest that APUM23 plays an important role in plant development via rRNA processing and ribosome biogenesis. To identify the genes affected in apum23-1 as compared to wild-type (Col-3) under normal growth conditions using Arabidopsis ATH1 Genome arrays (Affymetrix). RNA samples from these plants were used to generate biotin labelled (cDNA) probes, which were then hybridized to the microarray. For data set generation, three independent samples were used, three from Col-3 and three from apum23-1 plants grown on soil for 21 days.

Project description:T lymphocyte recognition of antigenic peptides on the surface of antigen-presenting cells (APC) leads to immune synapse formation (IS). By analogy with the nervous synapse, this cell-cell communication model requires the formation of highly organized structures that delimit an active zone of membrane traffic. For T cells to connect antigen recognition with the biological response, receptors and signaling proteins must be recycled. Sorting nexin 27 (SNX27) is an endosomal protein best known for its role in recycling PDZ (PSD95, Dlg1, ZO-1)-interacting neuronal proteins via the retromer transport machinery. Alteration of this sorting pathway leads to neurodegeneration and is associated to Down syndrome, Alzheimer’s and Parkinson’s diseases. In T lymphocytes, SNX27 interacts with diacylglycerol kinase zeta (DGKz), which provides a mechanistic link between diacylglycerol metabolism and membrane trafficking. Following antigen recognition, SNX27-positive endosomes polarize to the IS and a PDZ-dependent SNX27 pool accumulates at the cell-cell contact area. To test for additional SNX27 functions, we carried out proteomic analysis of the SNX27 interactome purified from T cells in contact with APC, and identified SNX27 interaction with the retromer and with members of the WASH (Wiskott-Aldrich syndrome protein and SCAR homologue) complex. This finding indicates the conserved nature of the SNX27/WASH/retromer complex in hematopoietic cells and suggests similarity between neurological diseases and abnormal immune/inflammatory responses. We also found PDZ-dependent SNX27 cargoes, including modulators of cytoskeletal reorganization such as zona occludens-2 (ZO-2). ZO-2, a cytosolic component of epithelial cell-cell junctions, localized to the IS, and SNX27 silencing affected ZO-2 stability at the synapse. This study broadens our knowledge of SNX27 function in T lymphocytes, and suggests that pathways that delimit polarized structures in epithelial systems also participate in IS regulation.

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Development of Automated Proteomic Workflows Utilizing Silicon-Based Coupling Agents

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