Dataset DOI: 10.5061/dryad.866t1g25g

Description of the data and file structure

Hydrogen-exchange mass spectrometry

A hydrogen-deuterium exchange mass spectrometry (HDX-MS) dataset, characterizing the binding of nanobodies to deglycosylated uPAR, is described. Briefly, deglycosylated uPAR was prepared by overnight incubation at 37°C with an enzyme cocktail (PNGaseF, neuraminidase, ß-galactosidase and N-acetylglucosaminidase). VHH:uPAR complexes were prepared at a 1:1 molar ratio in PBS, pH 7.4. Using an HDx3-PAL autosampler (Trajan Scientific and Medical), samples were labeled for 3 min by 5-fold dilution in labelling solution (90% D2O in PBS, pH 7.4) for a final deuterium composition of 72%, quenched for 2 min at 4°C by 5-fold dilution in 2 M urea containing 200 mM TCEP and 0.1% formic acid, then 75 µL (23 pmol) was injected onto an immobilized pepsin column at 10°C. Peptides were trapped and washed (ACQUITY UPLC BEH C18 VanGuard Pre-column, 130 Å, 1.7 µm, 2.1 mm × 5 mm, Waters) at a flow rate of 50 µL/min for 5 min, separated using a 1-35% acetonitrile gradient over 8 min (ACQUITY UPLC BEH C18, 130 Å, 1.7 µm, 1 × 100 mm, Waters), and analyzed using a SYNAPT G2-Si mass spectrometer (Waters). Peptide lists were generated using unlabeled samples collected with data-dependent acquisition, followed by database searching with Mascot. Data were collected in triplicate, and deuteration was assigned using MS Studio. Significant changes in deuteration were determined based on thresholds of 3× SD and 1-p = 0.95.

This dataset allows for insights into the conformational response of uPAR upon nanobody binding, and confirmed unique deuterium uptake profile of uPAR when complexed with XUp09 compared to XUp10 and XUp12. This corroborates the binding bins described by surface plasmon resonance experiments. 

Immunoprecipitation and mass spectrometry for anti-uPAR sdAbs

Immunoprecipitation experiments were conducted by incubating biotinylated VHH-loaded DynabeadsTM M-280 streptavidin beads (Thermo Fisher Scientific) with cleared and filtered HEK293-6E/h-UPAR or HEK293-6E parental cell lysate on ice for 1 h, washing, and elution with 8 M urea as described105. Immunoprecipitates were analyzed by SDS-PAGE and western blotting using serum from the uPAR ECD-immunized llama (1:5000) followed by HRP-conjugated anti-llama IgG antibody (100 ng/mL; Cedarlane, Cat#A160-100P). Immunoprecipitated bands were excised from SDS-PAGE gels, de-stained with 30% acetonitrile in 100 mM ammonium bicarbonate (AMBIC), dehydrated with 100% acetonitrile, reduced with 10 mM DTT, alkylated with 55 mM iodoacetamide, dehydrated, and then resuspended in trypsin (20 ng/μL in 50 mM AMBIC) overnight at 37°C. Peptides were collected and analyzed by C18 nanoLC-MS on an Orbitrap Exploris 240 mass spectrometer (Thermo Fisher Scientific) using a 72 min data-dependent acquisition method. MS2 spectra were searched against the Uniprot human database using MSFragger implemented in Fragpipe with a 1% false discovery rate. Peptides were quantified based on the MS1 ion intensity within 5 ppm and 50 scans of all identified m/z ions.

Files and variables

File: Immunoproteomics_data.zip

Description: 

The dataset contains 9 raw data files and associated processed data files. The raw files can be opened using Xcalibur; the MS acquisition parameters used are available within these files. The associated files include the mzML files used in the Fragpipe analysis, the calibrated mgf files generated by Fragpipe, and the pepXML result files generated by Fragpipe. Many libraries and tools exist to allow the parsing and use of these files. The file names of all processed files are named the same as the associated raw file. Each raw data file corresponds to a DDA analysis of a single gel band from the immunoprecipitated samples. The relationship between the gel band and the raw file is shown in the IP_SampleInfo.xlsx file, where sample names correspond to the gel image file, where the extracted gel bands are labelled (“GelImage-IP.pdf”). Each lane in the gel represents a different IP sample: (1) the resin alone (negative control; “Resin”), (2) immunoprecipitation with a VHH that recognizes a non-human antigen (“A20.1”), and (3) immunoprecipitation with the uPAR VHH (“Xup10”). Three bands were extracted from each lane, and identification results were combined by Fragpipe by putting all bands from the same sample into the same experimental group.

File: HDX_MS_data.zip

Description: 

The dataset includes the data-dependent mapping sample, and triplicate samples at a single labelling timepoint for each of the protein states included in the differential HDX-MS experiment. The .raw files (generated on a Waters Synapt G2Si) can be opened and viewed using either MassLynx (v4.1 or later, proprietary),  SeeMS from the open-source Proteowizard suite. (http://www.proteowizard.org/index.html), or Skyline (v26.1 https://skyline.ms/project/home/software/Skyline/begin.view?). These files can be imported into proprietary HDX-MS platforms such as Mass Spec Studio (Trajan Scientific), DynamX (Waters). Alternatively, deuterium can be calculated using open source software such as “The Deuterium Calculator” (https://pubs.acs.org/doi/10.1021/acs.jproteome.2c00558).

Each .raw folder acts as a container for the entire analytical run (mandatory files), and contains the following files. The entire folder must be opened by the software of choice to view files:

• _HEADER.TXT: Contains metadata such as date and vial position.
• _FUNCTNS.INF: Contains information about the functions (functions are typically MS experiments/scans).
• _FUNCn.DAT: Contains raw MS data for each function \(n\)
• _FUNC[X].IDX: Index files that allow the software to quickly locate specific scans within the corresponding .DAT binary files.
• _ FUNC0n.STS: status logs that track the progress and completion state of data acquisition to ensure the software can correctly verify and resume the run

A full list of the data files and structure is described in the file named: HDX_MS_sample_list.xlsx.

The user can generate a peptide list from the undeuterated sample by searching against the include protein list (uPAR_protein_database.fasta). Otherwise, the use can import the included peptide list (UPAR_2min_peptide_list_and_search_parameters.csv) into any HDX-MS software to interpret the raw data. For extracting differential HDX-MS data, the unbound deglycosylated uPAR samples should be used as the reference state, and the VHH-states as the bound samples. Differential deuteration is calculated by subtracting the deuteration of the unbound state from the bound state. The processed deuteration measurements (UPAR_MS_Studio_results.csv) results are included, should the user wish to directly inspect the processed output from MS Studio (the HDX-MS processing software used in this study).

The column headers in UPAR_MS_Studio_results.csv are as follows:

• Protein state: Name of the sample
• Start: Sequence number of the first amino acid in the peptide
• Stop: Sequence number of the last amino acid in the peptide
• Sequence: Amino acid sequence of the identified peptide
• Charge: Peptide charge state
• Peptide mass (Da): Monoisotopic peptide mass in Daltons
• Retention time (min): Chromatographic retention time in minutes
• HDX time (min): Experimental labelling time in minutes
• Uptake (D): Mean absolute deuteration uptake measured in Daltons (triplicate)
• Uptake SD (D): Standard deviation of the mean absolute deuteration uptake measured in Daltons (triplicate)
• Uptake (%): Mean normalized deuteration uptake (triplicate)
• Uptake SD (%): Standard deviation of the mean normalized deuteration uptake (triplicate)

The relationships between the data files are described in two separate tables:

• Immunoproteomics: IP_SampleInfo.xlsx
• Hydrogen-deuterium exchange mass spectrometry: HDX_MS_sample_list.xlsx