Dataset Information

BIOSITe: A novel method for direct detection of site specific biotinylation

ABSTRACT: Proximity-dependent biotinylation methods, such as those mediated by BioID and APEX, are being widely adopted for studying in vivo protein-protein interaction dynamics and subcellular structures. However, these along with other biotin-based technologies are severely dependent on avidin family proteins for the capture of biotinylated proteins. While the extremely high affinity of the avidin-biotin interaction nearly guarantees the capture of biotinylated proteins from a sample, it also results in an inability to recover the key species of interest: the biotin-modified peptide. To overcome this limitation, we have developed a novel strategy that instead relies on an immunoaffinity-based capture of biotinylated peptides for downstream LC-MS/MS analysis. We refer to this approach as Biotinylation Site Identification Technology (BioSITe), since it enables the direct detection of site-specific biotinylation, which substantially increases the sensitivity and specificity of data offered by proximity-dependent biotinylation methods. Using isotopically labeled biotin, we also demonstrate that this method can be used for differential analysis. Overall, we anticipate this method to replace the current methods used for detection of biotinylation sites and other applications including those employing any molecule probe for enrichment of specific classes of proteins or post-translational modifications.

INSTRUMENT(S): Orbitrap Fusion ETD, Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)

TISSUE(S): Bone Marrow, Kidney

SUBMITTER: Akhilesh Pandey

LAB HEAD: Akhilesh Pandey

PROVIDER: PXD007862 | Pride | 2017-12-19

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	DK_082617_APEX_IMS_ALL_REPS_oxBP_justY_with_IMS_APEX_FASTA.msf	Msf
	DK_082617_APEX_NES_ALL_REPS_oxBP_justY_With_NES_APEX_FASTA.msf	Msf
	DK_APEX_IMS_REP1.raw	Raw
	DK_APEX_IMS_REP2.raw	Raw
	DK_APEX_IMS_REP3.raw	Raw

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Publications

BioSITe: A Method for Direct Detection and Quantitation of Site-Specific Biotinylation.

Kim Dae In DI Cutler Jevon A JA Na Chan Hyun CH Reckel Sina S Renuse Santosh S Madugundu Anil K AK Tahir Raiha R Goldschmidt Hana L HL Reddy Karen L KL Huganir Richard L RL Wu Xinyan X Zachara Natasha E NE Hantschel Oliver O Pandey Akhilesh A

Journal of proteome research 20171228 2

Biotin-based labeling strategies are widely employed to study protein-protein interactions, subcellular proteomes and post-translational modifications, as well as, used in drug discovery. While the high affinity of streptavidin for biotin greatly facilitates the capture of biotinylated proteins, it still presents a challenge, as currently employed, for the recovery of biotinylated peptides. Here we describe a strategy designated Biotinylation Site Identification Technology (BioSITe) for the capt ...[more]

PMID: 29249144

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Project description:Background: Cell surface proteins perform critical functions related to immune response, signal transduction, cell-cell interactions, and cell migration. Expression of specific cell surface proteins can determine cell-type identity, and can be altered in diseases including infections, cancer and genetic disorders. Identification of the cell surface proteome remains a challenge despite the development of several enrichment methods exploiting their biochemical and biophysical properties. Methods: We report a novel method for enrichment of proteins localized to the cell surface. We developed surface Biotinylation Site Identification Technology (sBioSITe) by adapting our previously published method for direct identification of biotinylated peptides. The primary amine groups of lysines on cell surface proteins in live PC3 cells in culture were first labeled with biotin with subsequent enrichment of biotinylated peptides with anti-biotin antibodies. The enriched peptides containing biotin were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Biotinylated peptides identified in triplicate were mapped to proteins. Results: By direct detection of biotinylated lysines using sBioSITe, we identified 5,851 peptides biotinylated on the cell surface derived from 1,409 proteins. 533 of these proteins have been previously shown or predicted to be localized to the cell surface, or secreted into the extracellular space. Importantly, several of the identified markers have known associations with prostate cancer and metastasis including CD59, epithelial growth factor receptor (EGFR), 4F2 cell-surface antigen heavy chain (SLC3A2) and Adhesion G protein-coupled receptor E5 (ADGRE5) thus highlighting the robustness of this method. Conclusions: Detection of biotinylation sites on cell surface proteins modified in vitro provides a reliable method for the identification of cell surface proteins. This method is an excellent tool that complements existing methods for the analysis of surface expressed proteins in qualitative as well as comparative studies.

Project description:Ubash3b, also known as suppressor of T-cell receptor signaling or Sts-1, is an ill-studied atypical tyrosine phosphatase with ubiquitin binding ability. In our previous study, we hypothesized that Ubash3b plays an inhibitory role in BCR-ABL signaling through binding and dephosphorylating BCR-ABL and its interactors. The Hantschel lab recently solved the crystal structures of the p210 PH and DH domains, which are absent in the p190 variant, and demonstrated that loss-of-function mutations in the PH domain altered BCR-ABL localization, thereby reducing the interaction between Ubash3b and p2104. Taken together, this suggests differential subcellular localization of Ubash3b as a mechanism by which it interacts more strongly with p201 as compared to p190. To better understand the global impact Ubash3b has on p210, its direct kinase substrates and proteins in its phosphotyrosine signaling network, we have taken an integrative approach by combining global phosphotyrosine profiling, proximity-dependent biotinylation (BioID) and total protein analysis to investigate p210 signaling upon Ubash3b knockdown (KD). The BioID system was used to characterize Ubash3b function in p210 signaling by examining its interactome. Importantly, in all of our BioID experiments, we employed a newly technique that we have recently developed, Biotinylation Site Identification Technology (BioSITe), which directly identifies biotinylated peptides thereby increasing the reliability of the identified interactors. Here, we additionally used short hairpin RNA (shRNA) interference and generated Ubash3b knock-down (KD) and non-targeting control shRNA lines in Ba/F3 BirA*-p210 cells. Ubash3b expression was reduced >90 % in the KD cells and had a substantial effect on global tyrosine phosphorylation and on the interactome of p210. Of the 1,421 unique tyrosine phosphorylation sites identified from 830 proteins, 379 sites (from 286 proteins) exhibited a substantial increase (ÃƒÂ¢Ã¢â‚¬Â°Ã‚Â¥2-fold) in tyrosine phosphorylation upon Ubash3b KD cells compared to control cells. To date, the interactome of Ubash3b has not been extensively investigated, however, some examination of Ubash3b in the context p210 signaling has been undertaken. We designed constructs of C-terminal BirA* tagged full length Ubash3b and a deletion mutant lacking the UBA and SH3 domains leaving only the phosphatase domain tethered to BirA*. A comparative analysis of the core interactors of p210 from previous studies and Ubash3b interactome from the current study revealed 36 proteins that interact with both p210 and Ubash3b.

Project description:In the past years several protocols for the proteomic profiling of plasma membrane proteins have been described. Nevertheless, comparative analyses have mainly focused on different variations of one approach [1-3]. To pave the way to high-performance differential plasma membrane proteomics, we compared sulfo-NHS-SS-biotinylation, aminooxy-biotinylation and surface coating with silica beads to isolate plasma membrane proteins for subsequent analysis by one-dimensional gel-free liquid chromatography mass spectrometry. Absolute and relative numbers of plasma membrane proteins and reproducibility parameters on a qualitative and quantitative level were assessed. Sulfo-NHS-SS-biotinylation outperformed aminooxy-biotinylation and surface coating using silica beads for most of the monitored criteria. We further simplified this procedure by introducing a competitive biotin elution strategy, for which the average plasma membrane annotated protein fraction amounted to 54 % (347 proteins). Moreover, purified non-plasma membrane annotated proteins and plasma membrane annotated proteins displayed similarly high reproducibility suggesting specific co-purification. In fact, the non-plasma membrane annotated data was extremely enriched for direct interactors of purified plasma membrane proteins. Computational analysis using additional databases and prediction tools revealed that in total over 90 % of the purified proteins were associated with the plasma membrane. The modified sulfo-NHS-SS-biotinylation protocol was validated by tracking changes in the plasma membrane proteome composition induced by genetic alteration and drug treatment. GPI-anchored proteins were depleted in plasma membrane purifications from cells deficient in the GPI transamidase component PIGS; and treatment of cells with tunicamycin significantly reduced the abundance of N-glycoproteins in surface purifications. Altogether, this study introduces an improved, filter-free sulfo-NHS-SS-biotinylation protocol and demonstrates it to be a specific, effective and reproducible method to isolate proteins associated with the plasma membrane, thus enabling future large-scale comparative cell surface mappings.