Dataset Information

Biotinylation by antibody recognition - A novel method for proximity labeling

ABSTRACT: Identification of protein-protein interactions is a major goal of biological research. Despite technical advances over the last two decades, important but still largely unsolved challenges include the high-throughput detection of interactions directly from primary tissue and the identification of interactors of insoluble proteins that form higher-order structures. We have developed a novel, proximity-based labeling approach that uses antibodies to guide biotin deposition onto adjacent proteins in fixed cells and primary tissues. We showed our method to be specific and sensitive by labeling a mitochondrial matrix protein. Next, we used this method to profile the dynamic interactome of lamin A/C in multiple cell and tissue types under various treatment conditions. Our results suggest a considerable variation in the composition of the nuclear envelope of different tissues. Of note, DNA damage response proteins Ku70 and Ku80 are more abundant in the vicinity of lamin A/C after thermal stress. The ability to detect proximal proteins and putative interactors in intact tissues, and to compare affinities quantitatively under different conditions or in the presence of disease mutations, can provide a new window into cell biology and disease pathogenesis.

INSTRUMENT(S): Orbitrap Fusion Lumos, Orbitrap Fusion, LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Skeletal Muscle, Primary Cell, Adipose Tissue, Smooth Muscle, Hela Cell

SUBMITTER: Daniel Bar

LAB HEAD: Francis Collins

PROVIDER: PXD004736 | Pride | 2017-10-17

REPOSITORIES: Pride

ACCESS DATA

Publications

Biotinylation by antibody recognition-a method for proximity labeling.

Bar Daniel Z DZ Atkatsh Kathleen K Tavarez Urraca U Erdos Michael R MR Gruenbaum Yosef Y Collins Francis S FS

Nature methods 20171218 2

The high-throughput detection of organelle composition and proteomic mapping of protein environment directly from primary tissue as well as the identification of interactors of insoluble proteins that form higher-order structures have remained challenges in biological research. We report a proximity-based labeling approach that uses an antibody to a target antigen to guide biotin deposition onto adjacent proteins in fixed cells and primary tissues, which allows proteins in close proximity to the ...[more]

PMID: 29256494

Similar Datasets

Project description:Biomarkers for early detection of chronic kidney disease are needed, as millions of patients suffer from chronic diseases predisposing them to kidney failure. Protein microarrays may hold utility in the discovery of auto-antibodies in other conditions not commonly considered auto-immune diseases. We hypothesized that proteins are released as a consequence of damage at a cellular level during end-organ damage from renal injury, not otherwise recognized as self-antigens, and an adaptive humoral immune response to these proteins might be detected in the blood, as a non-invasive tracker of this injury. The resultant antibodies (Ab) detected in the blood would serve as effective biomarkers for occult renal injury, enabling earlier clinical detection of chronic kidney disease than currently possible, due to the redundancy of the serum creatinine as a biomarker for early kidney injury. To screen for novel autoantibodies in chronic kidney disease, 24 protein microarrays were used to compare serum Ab from patients with chronic kidney disease against matched controls. From a panel of 38 antigens with increased Ab binding, 4 were validated in 71 individuals, with (n=50) and without (n=21) renal insufficiency. Significant elevations in the titer of novel auto-Ab were noted against Angiotensinogen (AGT) and PRKRIP1 in renal insufficiency. Current validation is underway to evaluate if these auto-Ab can provide means to follow the evolution of chronic kidney disease in patients with early stages of renal insufficiency, and if these rising titers of these auto-Ab correlate with the rate of progression of chronic kidney disease. Serum antibodies were profiled for 7 healthy individuals and 17 patients with chronic kidney disease, using the Invitrogen ProtoArray® Human Protein Microarray v3.0 platform (Invitrogen, Carlsbad, CA). This platform contains 5,056 non-redundant human proteins expressed in a baculovirus system, purified from insect cells and printed in duplicate onto a nitrocellulose-coated glass slide. Each protein is spotted twice on each array, to measure the quality of the signal intensity. Details for experiment processing and analysis follow the previous publication from our group (Li et al Proc Natl Acad Sci U S A. 2009 Mar 17;106(11):4148-53). Prospector software was used to retrieve the expression based on immune response profiling of the .gal files.

Project description:Microarray technology has evolved as a powerful tool over the last decade, to identify biomarkers and study the mechanisms of diseases. We propose a novel application of integrated genomics by combining transcriptional levels with serological antibody profiling after kidney transplantation, with the aim of uncovering the relative immunogenicity of seven different renal compartments after allo-transplantation. Thirty-six paired pre- and post-transplant serum samples were examined from eighteen transplant recipients, across 5,056 protein targets on the ProtoArray V3.0 platform. Normal renal compartment-specific gene expression data from a cDNA platform were re-analyzed and both the cDNA and the ProtoArray platforms were re-annotated to most up-to-date NCBI gene identifiers; 3,835 genes/proteins are measured on both platforms. Antibody levels were ranked for individual patients and the hypergeometric enrichment statistic was applied on mapped compartment-specific expression data. We discovered that after transplantation, in addition to HLA and MICA responses, temporal alloimmune responses are seen against non-HLA antigens specific to different compartments of the kidney, with highest level responses noted against renal pelvis and cortex specific antigens. The renal medulla is of low immunogenicity as none of the outer or inner medulla specific targets generated significant post-transplant antibody responses. Immunohistochemistry confirmed pelvis and cortex specific localizations of selected targeted antigens, supporting the robust nature of this discovery. This study provides a road map of renal compartment-specific non-HLA antigenic targets responsible for generating alloimmune responses, opening the door for clinical correlations with post-transplant dysfunctional states to be determined. Keywords: alloimmune response after kidney transplantation Plasma profiling using Protein Microarray: Serum antibodies were profiled using Invitrogen ProtoArrayÂ® Human Protein Microarray v3.0 technology (Invitrogen, Carlsbad, CA). This platform contains 5,056 non-redundant human proteins expressed in a baculovirus system, purified from insect cells and printed in duplicate onto a nitrocellulose-coated glass slide. Five mL serum diluted in PBST buffer at 1:150 was applied for 90 minutes onto the Protoarray, after blocking with blocking buffer for 1 hour. The slides were then washed with 5ml fresh PBST buffer, 4 times for 10 minutes each, and probed with secondary antibody (goat anti-human Alexa 647, Molecular Probes, Eugene, OR) for 90 minutes. Finally, after a second washing with PBST buffer, the slides were dried and scanned using a fluorescent microarray scanner (GSI Luminoics Perkin-Elmer scanner). All steps were carried out on a rotating platform at 4 ÂºC. ProtoArray data acquisition and measurement: The slides were scanned at a PMT gain of 60% with a laser power of 90% and a focus point of 0 Î¼m. Fluorescence intensity data were acquired using GenePix Pro 6.0 software (Molecular devices, Sunnyvale, CA) with the appropriate â.galâ file downloaded from the ProtoArray central portal on the Invitrogen website (http://www.invitrogen.com/protoarray) by submitting the barcode of each ProtoArray slide.

Project description:Background: Studies recently support that non-HLA antigens could be additional targets of injury in organ transplant recipients, and MICA was associated with an increased risk of graft loss. Methods: A ProtoArray platform was used to study 37 serum samples from 22 unique patients (15 renal recipients and 7 healthy controls). Thirty paired pre- and post-transplant serum samples were analyzed for detection of de novo post-transplant antibody responses in the 15 patients (10 acute rejection (AR), 5 Stable). Probes on ProtoArray and cDNA platforms (GSE: 3931) were re-annotated and compartment specific gene lists were analyzed using the integrated genomics method. Normal and transplant kidney IHC were performed for MICA antigen localization. Results: Mean MICA-Ab (antibody) signal intensity was significantly higher in transplant patients compared with healthy controls and de novo MICA-Ab were detected in 73% transplant patients. The mean post-transplant signal intensity of MICA-Ab was the highest in C4d+AR. Detection of MICA-Ab responses did not correlate with time post-transplantation, but significantly correlated with decline in graft function over the subsequent year. Integrative genomics predicted localization of the MICA antigen to the glomerulus. IHC confirmed cytoplasmic MICA staining solely in glomerular podocytes in normal kidney. In the transplant kidney, infiltrating mononuclear lymphocytes (T, B and NK) in AR had additional MICA staining. Conclusions: MICA can be highly detected regardless of graft dysfunction or AR. The intensity signal of the MICA antibody correlates with subsequent decline in graft function. The MICA antigen localizes to the glomerulus and infiltrating mononuclear cells in AR. Pre- and post-transplant serum antibodies were profiled for each patient, using the Invitrogen ProtoArrayÂ® Human Protein Microarray v3.0 platform (Invitrogen, Carlsbad, CA). This platform contains 5,056 non-redundant human proteins expressed in a baculovirus system, purified from insect cells and printed in duplicate onto a nitrocellulose-coated glass slide. Each protein is spotted twice on each array, to measure the quality of the signal intensity. Details for experiment processing and analysis follow the previous publication from our group (13). Prospector software was used to retrieve the expression based on immune response profiling of the .gal files.

Dataset Information

Biotinylation by antibody recognition - A novel method for proximity labeling

Publications

Biotinylation by antibody recognition-a method for proximity labeling.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure