2.2. Preparation of Soluble Mating Secretome
In preliminary experiments, CC124 and CC125 gametes checked for high mating efficiency were mixed together for 1 h with gentle aeration at 22 °C. Following removal of cells (1600× g for 5 min) and debris (20,000× g for 10 min), the mating medium was filtered through a 0.22 µm filter to remove particulate material. The filtrate was concentrated 7-fold using a tangential flow filter system with a 2 kDa cutoff (Sartorius Vivaflow 200, Hydrosart, 2K; Thermo Fisher Scientific, Waltham, MA, USA). The concentrated mating medium was subjected to SDS-PAGE; after visualization by silver staining (Pierce, SilverSnap kit; Thermo Fisher Scientific, Rockford, IL, USA), the gel lane was excised. Gel fragments containing high and low molecular weight proteins were separately subjected to in-gel trypsin digestion, essentially as described below. Tandem mass spectrometry was carried out at the Proteomics and Mass Spectrometry Facility at the University of Massachusetts Medical School. The sample analyzed in this preliminary experiment contained both ectosomes and soluble proteins; in subsequent analyses, additional centrifugation steps yielded ectosomes and the soluble secretome, which were analyzed separately.
Gametes of both mating types (plus and minus) were washed and resuspended in 10 mL of fresh nitrogen-free medium at a density of 8–10 × 106 cells/mL. An equal number of mating type plus and minus gametes were mixed; after a 1 h incubation, the cultures were centrifuged at 1600× g as described above. These supernatants were then centrifuged at 20,000× g for 30 min at 4 °C to pellet cell debris. The resulting supernatants were next centrifuged at 200,000× g for 60 min at 4 °C to sediment all particulate material, including extracellular vesicles. These final supernatants are referred to as the soluble mating secretome. A protease inhibitor cocktail (Roche, cOmplete ULTRA Tablets, Cat. No. 05 892 791 001) and phenylmethylsulfonyl fluoride (final concentration 0.3 mg/mL) were added to each sample, which was dispensed in aliquots that were stored at −80 °C. A total of six samples prepared at two different times were subjected to analysis, ultimately yielding Dataset 1 (Samples A, B and C) and Dataset 2 (Samples D, E and F). SDS-PAGE fractionation of 40 μL of the soluble mating secretome yielded bands that were readily visualized using silver staining (Silver Stain for Mass Spectrometry; Thermo Fisher Scientific, Rockford, IL, USA). The low speed pellet (1600× g for 5 min) was resuspended in 0.30 mL 20 mM 2-[tris(hydroxymethyl)-methylamino]-ethanesulfonic acid (TES), 10 mM mannitol, pH 7.4 containing 1% TX-100 and a protease inhibitor cocktail; aliquots were assayed for protein content using the bicinchoninic acid assay (Thermo Fisher Scientific, Rockford, IL, USA).
2.3. Fractionation for Mass Spectroscopy
Aliquots (40 µL) of each soluble mating secretome were prepared for SDS-PAGE by mixing with 4× Laemmli sample buffer (Bio-Rad, Hercules, CA, USA) and denaturation at 55 °C for 5 min. Each sample was then fractionated on a Criterion TGX 4–15% gradient gel (Bio-Rad, Hercules, CA, USA). Electrophoresis of the first set of samples was stopped when the dye band had traveled 3 cm, and the gel was stained with QC Colloidal Coomassie (Bio-Rad, Hercules, CA, USA), according to the manufacturer’s protocol. Based on molecular weight standards analyzed at the same time, each lane was cut into 10 slices that covered material migrating from the dye band to the top of the gel. Electrophoresis of the second set of samples was stopped when the dye band had traveled 2.5 cm; after staining with QC Colloidal Coomassie, each lane was cut into 4 slices covering the entire molecular weight range. Gel slices were stored frozen in microfuge tubes before preparation for LC-MS/MS analysis after in-gel digestion with trypsin.
2.5. Mass Spectrometry
Gel bands were cut into small pieces, washed with 250 µL of 50% acetonitrile for 5 min with rocking, then washed with 50% acetonitrile/50 mM NH4HCO3 for 30 min on a tilt-table. After a final 30 min wash with 50% acetonitrile/10 mM NH4HCO3, gel fragments were dried using a speed vacuum. Each sample was suspended in 30 µL of 10 mM NH4HCO3 containing 0.20 µg of digestion grade trypsin (Promega, V5111) and incubated at 37 °C for 16 h. The digestion supernatant was acidified and placed into a vial for LC-MS/MS analysis (5 µL injected).
Data acquisition was conducted on an Orbitrap Fusion Tribrid mass spectrometer. Reversed phase (RP)-LC-MS/MS was performed using a nanoACQUITY UPLC system (Waters Corporation, Milford, MA, USA) connected to an Orbitrap Fusion Tribrid (Thermo Fisher Scientific, San Jose, CA, USA) mass spectrometer. After injection, samples were loaded into a trapping column (nanoACQUITY UPLC Symmetry C18 Trap column, 180 µm × 20 mm) at a flowrate of 5 µL/min and separated using a C18 column (nanoACQUITY column Peptide BEH C18, 75 µm × 250 mm). The compositions of mobile phases A and B were 0.1% formic acid in water and 0.1% formic acid in acetonitrile, respectively. Peptides were eluted with a gradient extending from 3% to 20% mobile phase B in 85 min, and then to 35% mobile phase B in another 35 min at a flowrate of 300 nL/min and a column temperature of 37 °C. The data were acquired with the mass spectrometer operating in a top speed data-dependent acquisition (DDA) mode. An Orbitrap full MS scan was performed in the range of 300–1500 m/z at a resolution setting of 120,000 with an automatic gain control (AGC) target value of 4 × 105. Iterative isolation (with a 1.6 Thomson unit isolation window and minimum intensity threshold of 5 × 104) and fragmentation by higher-energy collisional dissociation of ions were carried out after the Orbitrap full MS scan. Ions were injected with a maximum injection time of 110 ms and an AGC target of 1 × 105.
Raw data were processed using Proteome Discoverer software (version 2.1, Thermo Fisher Scientific, San Jose, CA, USA). MS2 spectra were searched using Mascot (Matrix Science, London, UK), which was set up to search against the Chlamydomonas reinhardtii
database (Creinhardtii_281_v5.5). The search criteria included 10 ppm precursor mass tolerance, 0.02 Da fragment mass tolerance, trypsin enzyme, and maximum missed cleavage sites of two. Dynamic modifications included propionamide on cysteine, oxidation on methionine, deamidation on asparagine and glutamine, and Gly-loss+Amide on C-terminal glycine. Peptide spectral match (PSM) error rates were determined using the target-decoy strategy coupled to Percolator modeling of true and false matches [23
]. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD010945.
Scaffold (version Scaffold_4.8.4, Proteome Software Inc., Portland, OR, USA) was used to validate MS/MS-based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 95.0% probability by the Scaffold Local FDR algorithm. Protein identifications were accepted if they could be established at greater than 99% probability and contained at least 1 identified peptide. Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. Proteins sharing significant peptide evidence were grouped into clusters.
A total of 1291 proteins were identified in Dataset 1 and in Dataset 2. Proteins recognized in less than four of the six samples were eliminated, yielding a Merged Dataset with 1216 proteins. We used PredAlgo (https://giavap-genomes.ibpc.fr/predalgo/
) to predict the subcellular localization of each identified protein. We used SignalP 4.1 (www.cbs.dtu.dk/services/SignalP/
) to identify proteins categorized as O (Other) or NA (Not Assigned) but predicted to contain a signal peptide (SP). The merged dataset contained 102 signal peptide-containing proteins, which we refer to as the soluble mating secretome.
2.6. Bioinformatic Analyses
To search for any neuropeptide-like precursors encoded by the C. reinhardtii
genome, we identified primary transcripts (Phytozome v5.5; a total of 17,741) encoding proteins with a predicted signal peptide (SignalP v4.1) and no transmembrane helices (TMHMM v2.0), resulting in a list of 771 proteins. Using a custom script, these protein sequences were searched for potential prohormone convertase cleavage sites ((K/R)Xn
(K/R), where n
= 0, 2, 4 or 6); likely furin cleavage sites (RX(K/R)R) were screened for separately. The cleavage sites identified were then screened for the presence of potential amidation sites (G(K/R)(K/R), (K/R)Xn
G(K/R) where n
= 1 or 3, and RG(K/R)R). In addition, NeuroPred [5
] was used with “known motifs” to predict amidated product peptides that could be produced from these proteins; based on cleavages observed in Aplysia californica
, RK, KXXK, and KXXR sites were excluded from “known motifs”. Multiple sequence alignments utilized T-COFFEE, Version 11.00 [25