Protein interactors of Spindle Pole Body (SPB) components and septal proteins in fungus Neurospora crassa: A mass spectrometry-based dataset

Microtubule Organizing Centers (MTOC) are subcellular structures in eukaryotic cells where nucleation of microtubules (MTs) takes place and represents the filament's minus end. Their localization depends on the species, cell type, and cell cycle stage. Along the fungal kingdom, the Spindle Pole Body (SPB) in the nucleus (an equivalent to Centrosomes in animal cells) is the principal MTOC. Other MTOCs have been identified in filamentous fungi, such as the Spitzenkörper in the hyphal tips of Schizosaccharomyces pombe or the septal pore of Aspergillus nidulans. However, in the fungal-model organism Neurospora crassa, these alternative MTOCs have not been recognized. Here, we present a Mass spectrometry-based dataset of proteins interacting with four MTOC components of N. crassa tagged with fluorescent proteins: γ-Tubulin-sGFP (main nucleator at the SPB), MZT-1-sGFP (structural SPB microprotein), APS-2-dRFP (septal protein and recognized SPB component), and SPA-10-sGFP (septal MTOC protein). A WT and a cytosolic GFP expressing strain were included as controls. The protein interactors were pulled down by Co-IP1, using GFP-Magnetic agarose that captures recombinant GFP proteins (including GFP-derivatives) in their native state. Bounded proteins were separated by SDS-PAGE and identified by nano LC-MS/MS2. The protein annotation was done using the N. crassa protein database.

strain were included as controls.The protein interactors were pulled down by Co-IP 1 , using GFP-Magnetic agarose that captures recombinant GFP proteins (including GFP-derivatives) in their native state.Bounded proteins were separated by SDS-PAGE and identified by nano LC-MS/MS 2 .The protein annotation was done using the N. crassa protein database.
© Instruments: Ultimate 30 0 0 nano UHPLC system with a trapping column (PepMap C18, 100 Å, 100 μm × 2 cm, 5 μm) and an analytical column (PepMap C18, 100 Å, 75 μm × 50 cm, 2 μm).Q Exactive HF mass spectrometer (Thermo Fisher Scientific, USA) with an ESI nanospray source.Protocol: Total native proteins from six strains of N. crassa were extracted and incubated with GFP-Magnetic agarose pearls to trap fluorescent-labeled proteins and their interactors.The beads were separated in a magnetic rack, and, after five washes, bound proteins were suspended in a 2x SDS-Sample buffer.Eluted proteins were electrophoresed in SDS-PAGE and stained with Coomassie blue.Selected gel lanes were cut and sent for protein identification service, where the in-gel digestion with trypsin, peptide extraction, desalting, and LC-MS/MS analysis were performed.The N. crassa database was used for protein annotation.Data

Value of the Data
• The raw data can be re-analyzed with a different method and search parameters to output new peptide search, protein inference, or protein concentration comparison.For example, the relative concentration of fusion proteins can be determined based on GFP quantification.• Results files contain the mass spectrometry report of identified peptides and proteins with their relative protein abundance in the sample (intensity) and a score (confidence of identified results).According to the protein identification service provider, a > 50 score denotes high-confident results.• Filtered lists display an extensive list of putative interactors ( > 10 0 0 per experiment) for γ -tubulin, MZT-1, APS-2, and SPA-10 proteins.Exploring these results opens the opportunity to explore novel physical interactions of those targets in vitro and in vivo .
• The whole dataset can be further exploited for researchers interested in studying MT assembly, SPB composition, or septal dynamics in N. crassa and other filamentous fungi.• The control experiments (WT and cytosolic GFP) can also detect spurious data in equivalent experimental conditions.

Data Description
Here, we present a data set of proteins and peptides identified by Mass Spectrometry after Co-IP experiments.The Co-IP was carried out using native protein extracts of six recombinant N. crassa strains incubated with GFP-Trap Magnetic agarose.The files with the raw extension are MS data obtained by Nano LC-MS/MS Analysis.The Excel files contain the report of the characterized peptides using the N. crassa protein database.
• 1_WT.rawfile provides data from a negative control consisting of native proteins of a WT strain (FGSC 4200) bound to GFP-Trap magnetic beads under native conditions.• 2_GFP.rawfile provides MS data from a positive control consisting of sGFP bound to GFP-Trap magnetic beads under native conditions.The crude extracts of the N. crassa strain expressing the sGFP in the cytosol were used for this control.

Experimental Design, Materials and Methods
Protein-protein interactions of γ -tubulin (main MTOC component), MZT-1 (integral MTOC microprotein), APS-2 (MTOC and septal protein), and SPA-10 (pore septal protein) were pull-down by Co-IP and identified by LC-MS/MS using the experimental design depicted in Fig. 1 .Here, a cytosolic sGFP expressing strain was included as a positive control and a WT strain as negative for background subtraction.
• L-Histidine stock solution: 1.25 g Histidine in distilled water to a final volume of 50 mL and sterilized through a 0.45 μm membrane.• Fixing solution (500 mL): 250 mL ethanol, 50 mL glacial acetic acid, and 200 mL distilled water.• Pre-stained protein marker.
• Ultimate 30 0 0 nano UHPLC system coupled with a Q Exactive HF mass spectrometer (Thermo Fisher Scientific, USA) with an ESI nanospray source.

Strains phenotype confirmation
All strains described in Table 1 were first grown from conidia stocks in VMM agar plates, except the TRM-129-RR11 strain (expressing SPA-10-sGFP), which was grown in VMM agar supplemented with histidine (500 μg/mL) and hygromycin (300 μg/mL).The agar plates were incubated at 30 °C in the dark for 12-16 h.After that, a piece of agar with mycelium was cut to check the fluorescence by confocal microscopy by the inverted agar block method [1] .A drop of 5 μl FM4-64 dye (5 μM) was placed to visualize the cell membrane.

Biomass Production
Once the fluorescence was verified, all six strains were inoculated from conidia stocks (10 μL) in 50 mL of liquid VMM medium into 250 mL flasks.The flasks were covered with aluminum, and the cultures were incubated for three days at 30 °C with constant shaking (150 rpm).In the end, the biomass was collected on filter papers using a vacuum pump (without washing).Mycelia were stored at −80 °C or used immediately.

Protein Extraction
The mycelia were ground with liquid nitrogen in sterile mortars until a fine powder was obtained.The shredded biomass was weighed in a fresh 15 mL centrifuge tube, and ice-cold extraction buffer was added in 1:1 proportion (w/v) ( e.g. , 1 mL buffer by 1 g biomass).The suspension was gently mixed by pipetting up and down to reduce the mechanical damage to a minimum.The insoluble material was collected by centrifugation at 12,0 0 0 rpm, 10 min, and 4 °C.After that, the supernatant was collected with a syringe and clarified through a 0.22 μm syringe filter.Aliquots of 100 μL were made to check protein quality and quantity, and the crude extracts were stored at −20 °C.

Co-IP
Once the quantity and quality of the extracts were confirmed, the native proteins (input) were incubated with GFP-Magnetic agarose (GFP monoclonal nanobody/ VHH conjugated to magnetic agarose beads).The beads were previously washed and equilibrated in the extraction buffer according to the manufacturer's instructions.The binding was carried on 1.5 mL tubes with 1 mL of protein suspension (4-6 mg) and 10-15 μL bead slurry.The mixture was incubated overnight at 4 °C, under constant mixing (on a rotator) at 4 °C.Afterward, the beads were separated in the magnetic rack, and the supernatant was removed and stored at −20 °C (labeled as non-bound).The beads were washed four times with 500 μl ice-cold wash buffer.In each step, aliquots were stored at 4 °C (labeled as wash 1-4).Once more, the beads were suspended in 500 μl ice-cold wash buffer and transferred to a new 1.5 mL fresh tube, followed by magnetic separation and supernatant discard (wash 5) ( Fig. 3 A depicts the Co-IP steps in a representative experiment).The binding material was suspended in 50 μl 2x SDS-Sample buffer and boiled for 5 min at 95 °C.The tube was spun for 1 min at 12,0 0 0 rpm.The elution (labeled as bound) was stored at 4 °C.

SDS-PAGE
Two biological replicates of Co-IP experiments were analyzed in SDS-PAGE 8% stained with Coomassie blue [7] .Analyzing bounded proteins was carried out in a laminar flow hood, using sterile fresh-prepared solutions and sterile gel electrophoresis glasses.Gel casts and plastic materials were adequately cleaned and UV-sterilized for 15 min after use.The gel bands with bound samples (2 per experiment) were aseptically cut with a scalpel and transferred into 15 mL new centrifuge tubes with 5 mL distilled sterile water.The samples were sent in an ice pack to Creative Proteomics (Shirley, NY) for protein identification service ( Fig. 3 B shows one lane of each sample).

In-gel digestion
Each gel slice was cut into cubes of 1 mm3 and transferred to 1.5 mL microcentrifuge tubes with 1 mL 50 mM NH 4 HCO 3 / acetonitrile (ACN) (1:1, v:v).After 30 min of incubation, the supernatant was removed, and the procedure was repeated until a complete discoloration.Next, 500 μL of ACN (Sigma-Aldrich) was added, and the mixture was incubated for 30 min.In the end, the gel pieces become opaque and stick together.ACN supernatant was removed, and the gel slices were rehydrated in 10 mM DL-dithiothreitol (DTT) (Sigma-Aldrich), followed by incubation at 56 °C for 1 h.The DTT was carefully removed, and 500 μL of ACN was added and incubated for 10 min at room temperature.The ACN was carefully removed, and 50 mM iodoacetamide (IAA) (Sigma-Aldrich) was poured until the gel slice was covered entirely.Then, an incubation period of 30 min at room temperature in the dark.The IAA was removed, and the gel slices were incubated in 500 μL of ACN for 10 min at room temperature.Next, the ACN solution was discarded, and a Rapid-Digestion Trypsin solution (Promega) was poured to cover the gel slices.The tube was incubated on ice for 45 min.If needed, more digestion solution was added if the gel pieces absorbed all the initial solution.The excess digestion solution was removed and added 5-20 μL of 50 mM NH 4 HCO 3 was to keep the gel pieces wet during enzymatic digestion.The excess digestion solution was removed, and 5-20 μL of 50 mM NH 4 HCO 3 was used to keep the gel pieces wet during enzymatic digestion.The supernatant was transferred into a fresh 1.5 mL microcen-trifuge tube where 50 mM ammonium bicarbonate/acetonitrile solution (1:2, v/v) was added to cover gel slices.An incubation period of 1 h was set at 37 °C.The solution was transferred to a new 1.5 mL microcentrifuge tube to lyophilize the extracted peptides to near dryness.Peptides were resuspended in 20 μL of 0.1% formic acid before LC-MS/MS analysis.

Nano-Liquid chromatography
For Liquid Chromatography, 1 μg of the sample was loaded and subject to a linear gradient from 2 to 8% buffer B for 3 min, then from 8% to 20% buffer B for 50 min; next, from 20% to 40% buffer B in 43 min; and finally, from 40% to 90% buffer B in 4 min, in the mobile phase A. The total flow rate was 250 nL/min.

Mass spectrometry
The full scan was performed between 300 and 1650 m/z at the resolution 60,000 at 200 m/z .The automatic gain control target for the full scan was set to 3e6.The MS/MS scan was operated in Top 20 mode using the following settings: resolution 15,0 0 0 at 200 m/z ; automatic gain control target 1e5; maximum injection time 19 ms; normalized collision energy at 28%; isolation window of 1.4 Th; charge sate exclusion: unassigned, 1, > 6; dynamic exclusion 30 s.

Data Analysis
The six raw MS files were analyzed and searched against the N. crassa protein database based on the species of the samples using MaxQuant (1.6.0.1) [8] .The detailed protein and peptide identification information was sent in an Excel file.
Approximately two-thirds of the identified proteins were shared with those listed in the controls (WT and sGFP).To subtract the background and potential spurious results, all the proteins listed in the controls were eliminated from the experiment.After that, more than 500 proteins remained for each experiment (Filtered lists).

Limitations
The methods described here apply to the Co-IP technique using magnetic agarose preimmobilized with anti-GFP antibodies (host alpaca), which lacks heavy and light antibody chains.Hence, it is not suitable for a free antibody IP approach.Also, a higher background is expected when using whole anti-GFP antibodies (with heavy and light antibody chains).

Ethics Statement
This work does not involve human subjects, animal experiments, or data collected from social media platforms.The authors have read and declare that the manuscript meets all the ethical requirements for publication (according to https://www.elsevier.com/authors/journal-authors/policies-and-ethics ).

Fig. 1 .
Fig. 1.Experimental design to determine the protein-protein interactions.Before each experiment, we activate all strains on Vogel's minimal medium (VMM) agar to verify the viability and fluorescence by Confocal Fluorescent Microscopy.The biomass was obtained in liquid VMM at 30 °C, 150 rpm for 3 days (in the dark).The mycelia were macerated in liquid nitrogen and suspended in an extraction buffer (1:1 w/v).The crude extracts were clarified by centrifugation and filtration through a 0.22 μm membrane, and the Bradford method determined the protein amount.Also, the protein integrity and fluorescence were analyzed on native PAGE electrophoresis.For Co-IP assays, 1 mL of native protein extracts were incubated with 10-15 μL of GFP-Magnetic agarose beads overnight at 4 °C.After five gentle wash steps using a magnetic rack, the bounded proteins were isolated and eluted in a 2x SDS-Sample buffer.Pulldown proteins were analyzed in SDS-PAGE, and gel bands were cut and stored in sterile distilled water at 4 °C.Two biological replicates per experiment were sent for protein identification service by LC-MS/MS.

Fig. 2 .
Fig. 2. Native protein extractions for Co-IP experiments.A) Protein quantification by the Bradford method.Dots denote the values of each protein extraction, and scripts represent the arithmetic mean.Color code indicates fluorescence: red is the dRFP tag, green is the sGFP, and gray is no fluorescence registered by native PAGE or microscopy.B) Visualization of protein profiles in native PAGE 10% using a fluorescence filter.C) Coomassie blue staining after fluorescence visualization.Codes: M molecular weight marker, 1 WT, 2 sGFP, 3 γ -Tubulin-sGFP, 4 MZT-1-sGFP, 5 dRFP-APS2, 6 SPA10-sGFP.
2024 The Authors.Published by Elsevier Inc.
glycerol, 1 mM PMSF, 1 mM Benzamidine, and protease inhibitor cocktail): 1.21 g Tris base, 0.58 g NaCl, 100 μL EDTA 0.5 M (pH 8.0), and 30 mL glycerol in 40 mL distilled water.Adjust pH with concentrated HCl solution, brought to a volume of 97 mL with distilled water, and sterilized through a 0.22 μM membrane.The working solution must be prepared at the moment by adding 10 μL PMSF (100 mM), 10 μL Benzamidine (100 mM), and 10 μL protease inhibitor cocktail per milliliter to be used.•

Table 1
List of neurospora crassa strains used in the experiments.