Protein Identification by Nanopore Peptide Profiling

This dataset belongs to “Protein Identification by Nanopore Peptide Profiling” and describes the raw data and analysis of tryptic digested peptides translocating through a mutant Fragaceatoxin C nanopore. A jupyter notebook describing the analysis and structure is added to this dataset. Data description: Protein Identification by Nanopore Peptide Profiling.ipynb Jupyter notebook contained data analysis of data contained in data_0.zip and data_1.zip (Python 3.7) python_scripts.zip Supplementary  scripts belonging to “Protein Identification by Nanopore Peptide Profiling.ipynb”. See explanation of custom classes in the jupyter notebook. data_0.zip - Folder containing raw electrophysiology data and result after analysis with “Protein Identification by Nanopore Peptide Profiling.ipynb”, with each folder containing the following: Alpha casein: Tryptic digest of alpha casein Beta casein: Tryptic digest of beta casein BSA: Tryptic digest of bovine serum albumin Control: Tryptic digest of water (no protein, control measurement) Cytochrome c: Tryptic digest of cytochrome c DHFR_His6: Tryptic digest of dihydropholate reductase (His6 tagged) EFP: Tryptic digest of elongation factor P HMW1Act: Tryptic digest of high molecular weight adhesin protein data_1.zip - Folder containing raw electrophysiology data, comma-separated MS peptide masses, and result after analysis with “Protein Identification by Nanopore Peptide Profiling.ipynb”, with each folder containing the following: Lysozyme: Tryptic digest of lysozyme               PAN: Tryptic digest of proteasome-activating nucleotidase TbpA_Y27A: Tryptic digest of periplasmic binding protein Trypsin: Tryptic digest of bovine trypsin Mass_spec: csv files containing measured ESI-MS peptides Lysozyme synthetic peptides: Synthetic peptides: Lys1: TPGSR Lys2alk:  C(+57.02)ELAAAMK Lys3: HGLDNYR Lys4alk: WWC(+57.02)NDGR Lys5: GTDVQAWIR Lys6alk: GYSLGNWVC(+57.02)AAK Lys7:       FESNFNTQATNR The structure of the data files is registered data_1.zip in 'index.csv' (digested proteins) and  'index_peptides.csv' (synthetic peptides) contained in the data folder. In this file, we describe the protein that was measured as well as the folder location and the expected baseline / standard deviation. Structure of ./data/index.csv Protein (string) | Folder (string) | Baseline (pA) (float) | Baseline Error (pA) (float) In each Folder, there is another 'index.csv', explaining which files are with protein and which are without (blank). Structure of ./data/[protein]/[repeat]/index.csv blank (boolean) | fname (string) Each folder in data_0.zip and data_1.zip contains a folder for each measure protein, which contains a folder for each repeat. The repeats contain raw axon binary files (.abf), each file contains measurement conditions as follows:     [Date of measurement]_[Pore type]_[Buffer conditions]_[added analyte(s)]_[operator initials]     e.g: 20200312_1M_KCl_50mM_Citricacid_50mM_BTP_pH_38_FraC_G13F_neg70mV_20ul_CytC_TrypsinGold_FL_0000     Measured on 12-03-2020, in 1M KCl buffered with Citricacid (50 mM) adjusted using bis-tris-propane to pH 3.8, using Fragaceatoxin C mutant G13F at a negatively applied potential of 70 mV. 20 µL cytochrome c was added to the cis compartment. The total volume of the container used for all electrophysiology experiments was 400 µL, all samples were prepared at a 1 g/L concentration. A prefix “perf” before analyte description indicates that the chamber was flushed with approximately 2 mL fresh buffer prior to analysis. The buffer condition "BTP" means bis-tris-propane, which is used to titrate to the exact pH of 3.8. Each analysed folder contains results.pkl file, containing the analysis result as provided by “Protein Identification by Nanopore Peptide Profiling.ipynb” - see the jupyter notebook Each analysed folder contains results_analysis.xlsx, which contains sheets with excluded currents, standard deviations, dwell time and beta value for the pore without analyte added “Blank” and results from the analyte added in “Results”. Parameters used for fitting are contained in “Parameters”. The “Histograms” tab shows the raw data of the excluded current spectra. mass_spec_peaks.zip – Folder containing mass spectrometry files as analysed by PEAKS Studio The folder contains an subfolder for each protein measured using electrospray ionisation mass spectrometry (ESI-MS). acasein: alpha casein protein b_casein: beta casein protein BSA: bovine serum albumin CytC: cytochrome C digested DHFR: dihydropholate reductase HMW1_Act: high molecular weight adhesin protein PAN: proteasome-activating nucleotidase ThBP: periplasmic thiamine binding protein Trypsin: bovine trypsi

CCDC 1454022: Experimental Crystal Structure Determination

Related Article: Niek N. H. M. Eisink, Martin D. Witte, Adriaan J. Minnaard|2017|ACS Catalysis|7|1438|doi:10.1021/acscatal.6b03459,An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.

CCDC 1917481: Experimental Crystal Structure Determination

Related Article: Federica Mancini, Anna K. H. Hirsch, Volker Huch|2019|CSD Communication|||,Related Article: Federica Mancini, M. Yagiz Unver, Walid A. M. Elgaher, Varsha R. Jumde, Alaa Alhayek, Peer Lukat, Jennifer Herrmann, Martin D. Witte, Matthias Köck, Wulf Blankenfeldt, Rolf Müller, Anna K. H. Hirsch|2020|Chem.-Eur.J.|26|14585|doi:10.1002/chem.202002250

CCDC 1917480: Experimental Crystal Structure Determination

Related Article: Federica Mancini, Anna K. H. Hirsch, Volker Huch|2019|CSD Communication|||,Related Article: Federica Mancini, M. Yagiz Unver, Walid A. M. Elgaher, Varsha R. Jumde, Alaa Alhayek, Peer Lukat, Jennifer Herrmann, Martin D. Witte, Matthias Köck, Wulf Blankenfeldt, Rolf Müller, Anna K. H. Hirsch|2020|Chem.-Eur.J.|26|14585|doi:10.1002/chem.202002250

CCDC 1917479: Experimental Crystal Structure Determination

Related Article: Federica Mancini, Anna K. H. Hirsch, Volker Huch|2019|CSD Communication|||,Related Article: Federica Mancini, M. Yagiz Unver, Walid A. M. Elgaher, Varsha R. Jumde, Alaa Alhayek, Peer Lukat, Jennifer Herrmann, Martin D. Witte, Matthias Köck, Wulf Blankenfeldt, Rolf Müller, Anna K. H. Hirsch|2020|Chem.-Eur.J.|26|14585|doi:10.1002/chem.202002250

Metabolic Labeling of Legionaminic acid in Flagellin Glycosylation of Campylobacter jejuni Identifies Maf4 as a Putative Legionaminyl Transferase

Campylobacter jejuni is the major human food-borne pathogen. Its bipolar flagella are heavily O-glycosylated with microbial sialic acids and essential for its motility and pathogenicity. However, both the glycosylation of flagella and the exact contribution of legionaminic acid (Leg) to flagellar activity is poorly understood. Herein, we report the development of a metabolic labeling method for Leg glycosylation on bacterial flagella with probes based on azide-modified Leg precursors. The hereby azido-Leg labeled flagellin could be detected by Western blot analysis and imaged on intact bacteria. Using the probes on C. jejuni and its isogenic maf4 mutant we also further substantiated the identification of Maf4 as a putative Leg glycosyltransferase. Further evidence was provided by UPLC–MS detection of labeled CMP-Leg and an in silico model of Maf4. This method and the developed probes will facilitate the study of Leg glycosylation and the functional role of this modification in C. jejuni motility and invasiveness

Metabolic Labeling of Legionaminic acid in Flagellin Glycosylation of Campylobacter jejuni Identifies Maf4 as a Putative Legionaminyl Transferase

Campylobacter jejuni is the major human food-borne pathogen. Its bipolar flagella are heavily O-glycosylated with microbial sialic acids and essential for its motility and pathogenicity. However, both the glycosylation of flagella and the exact contribution of legionaminic acid (Leg) to flagellar activity is poorly understood. Herein, we report the development of a metabolic labeling method for Leg glycosylation on bacterial flagella with probes based on azide-modified Leg precursors. The hereby azido-Leg labeled flagellin could be detected by Western blot analysis and imaged on intact bacteria. Using the probes on C. jejuni and its isogenic maf4 mutant we also further substantiated the identification of Maf4 as a putative Leg glycosyltransferase. Further evidence was provided by UPLC–MS detection of labeled CMP-Leg and an in silico model of Maf4. This method and the developed probes will facilitate the study of Leg glycosylation and the functional role of this modification in C. jejuni motility and invasiveness

CCDC 1845085: Experimental Crystal Structure Determination

Related Article: Ji Zhang, Niek N. H. M. Eisink, Martin D. Witte, and Adriaan J. Minnaard|2019|J.Org.Chem.|84|516|doi:10.1021/acs.joc.8b01949,An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.

CCDC 1853112: Experimental Crystal Structure Determination

Related Article: Ji Zhang, Niek N. H. M. Eisink, Martin D. Witte, and Adriaan J. Minnaard|2019|J.Org.Chem.|84|516|doi:10.1021/acs.joc.8b01949,An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.

CCDC 841692: Experimental Crystal Structure Determination

Related Article: G.Schaeffer, J.M.Harrowfield, J.-M.Lehn, A.K.H.Hirsch|2012|Polyhedron|41|40|doi:10.1016/j.poly.2012.04.013,An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.