DOI: 10.5061/dryad.stqjq2c5f
title: FASTA file of sequences of identified proteins in Anastrepha ludens reproductive tissues

description: Seminal fluid proteins (Sfps) modify female phenotypes and have
 wide-ranging evolutionary implications on fitness in many insects.
 However, in the Mexican fruit fly, Anastrepha ludens, a highly destructive
 agricultural pest, the functions of Sfps are still largely unknown. To
 gain insights into female phenotypes regulated by Sfps, we used nano
 liquid chromatography mass spectrometry to conduct a proteomic analysis of
 the soluble proteins from reproductive organs of A. ludens. The proteins
 predicted to be transferred from males to females during copulation were
 100 proteins from the accessory glands, 69 from the testes, and 20 from
 the ejaculatory bulb, resulting in 141 unique proteins after accounting
 for redundancies from multiple tissues. These 141 included orthologs to
 Drosophila melanogaster proteins involved mainly in oogenesis,
 spermatogenesis, immune response, lifespan, and fecundity. In particular,
 we found one protein associated with female olfactory response to
 repellent stimuli (Scribble), and two related to memory formation (aPKC,
 Shibire). Together, these results raise the possibility that A. ludens
 Sfps could play a role in regulating female olfactory responses and memory
 formation and could be indicative of novel evolutionary functions in this
 important agricultural pest.
description: <strong>Experimental design</strong>
 The proteomic analysis was performed using reproductive tissues
 from males and females. We focused on three tissues from males (MAG,
 testes, and ejaculatory bulb, Fig. 1a), whereas we used the lower
 reproductive tracts (without the ovaries and accessory glands) of females
 that were either mated or unmated (Fig. 1b). The method where
 non-chemically marked organs from both females and males are used have
 been used in other proteomic studies with success [13]. We analyzed a
 total of two replicates for MAG and reproductive tracts of unmated and
 mated females, and one replicate for the testes and ejaculatory bulb
 tissues. Each replicate (i.e., experimental unit) was comprised of tissues
 from 30 insects. <strong>Rearing and mating of
 flies </strong> Mass-reared pupae were obtained
 from Moscafrut, Metapa de Dominguez, Chiapas, Mexico. Adults were
 separated by sex after emergence and kept in controlled conditions (12:12
 h light: darkness cycle, 25 ± 2°C, 60 ± 10% RH). Three days after the
 emergence of the adults, males and females were separated and placed in
 cages, and fed sucrose and yeast hydrolysate (3:1 ratio). Matings were
 observed when adults were 15 days of age. In this species, males and
 females reach reproductive maturity at 10-14 days after emerging depending
 on their nutritional history [14, 15].
 <strong>Dissection of reproductive tracts
 </strong> Females were dissected within 10 min
 after mating ended, on a glass slide with wells filled with a buffer
 solution (10X PBS, 10% SDS & 7X Complete, Mini, EDTA-free Protease
 Inhibitor Tablet; Roche, Mannheim, Germany; every component was diluted to
 a 1X working solution) at 14 ± 2°C. Dissection and isolation of
 reproductive tracts were performed using a stereo microscope (SZX7 zoom;
 Olympus, Tokyo, Japan). To minimize protein degradation, dissections were
 carried out on cold gel blocks with sterilized tweezers (forceps # 5;
 Zwiss, Rubis, Switzerland). <strong>Protein
 extraction </strong> The tissues of <em>A.
 ludens</em> were processed and protein quantity was assessed using
 the BCA Assay Kit (Pierce, Rockford, IL). In brief, tissues were
 homogenized with 3 mL lysis buffer containing 150 mM Tris pH (8.2), 1%
 SDS, and 1 mM phenylmethylsulfonyl fluoride. Subsequently, the mixture was
 centrifuged at 10,000×g for 15 minutes, and the supernatant was recovered
 and stored at -80°C for further analysis. To remove the majority of sperm
 proteins from the analysis, the proteins that aggregated in the pellet
 were not analyzed. Thus, the majority of the identified proteins would be
 soluble seminal fluid proteins. <strong>Protein
 digestion</strong> A total of 50 µg of protein
 extract for all tissues was reduced with 10 mM Tris (2-carboxyethyl)
 phosphine for 45 min at 60°C, alkylated with 30 mM iodoacetamide for 1 h
 at 25°C and darkness, and then quenched the remaining of Iodoacetamide
 with 30 mM DTT for 10 min. Then, we precipitated proteins with four
 volumes of acetone at -20°C for 15 h. Proteins were then recovered by
 centrifugation at 3000×g for 30 min at 4°C. The supernatant was discarded,
 and the protein pellet was resuspended in 100 μL of 50 mM ammonium
 bicarbonate. Proteins were digested with trypsin (cat. No. V528A, Trypsin
 Gold, Promega, Madison, WI) at a 1:30 (w/w) trypsin protein ratio, for 16
 h at 37°C. Afterward, trypsin was added in 1:60 (w/w) trypsin protein
 ratio for 4 h at 3°C. Subsequently, proteins were fractionated using
 strong cation exchange cartridges (Thermo Scientific, Bellefonte, PA,
 USA). Protein fractionation resulted in three phases based on sequential
 elution with 150, 250, and 500 mM of KCl (elution buffer). Then, each
 fraction was desalted with C18 cartridges and dried using a CentriVap
 (Labconco; Kansas City, MO). <strong>Nano
 LC-MS/MS analysis</strong> Samples were analyzed
 by nano LC-MS/MS analysis using an Orbitrap
 Fusion<sup>TM</sup> Tribid<sup>TM</sup> mass
 spectrometer interfaced with an UltiMate 3000 RSLC system (Dionex;
 Sunnyvale, CA) and set with an “EASY Spray” nano ion source (Thermo-Fisher
 Scientific; San Jose, CA). Each reconstituted sample (5 μL) was loaded
 into a nanoviper C18 trap column (3 µm, 75 µm x 2 cm, Dionex) at 3 μL
 min<sup>-1</sup> flow rate and separated on an EASY spray C18
 RSLC column (2 µm, 75 µm × 25 cm), using a 100 min gradient with a flow
 rate of 300 nL min-1, and using 0.1% formic acid in LC-MS grade water
 (solvent A) and 0.1% formic acid in 90% acetonitrile (solvent B). The
 gradient was as follows: 10 min solvent A, 7-20% solvent B within 25 min,
 20% solvent B for 15 min, 20-25% solvent B for 15 min, 25-95% solvent B
 for 20 min, and 8 min solvent A. The mass spectrometer was operated in the
 positive ion mode with nano spray voltage set at 3.5 kV and source
 temperature at 280 °C. External calibrants included caffeine,
 Met-Arg-Phe-Ala (MRFA), and Ultramark 1621.
 <strong>Decision tree-driven MS/MS</strong>
 The mass spectrometer was operated in a data-dependent mode.
 Briefly, survey full-scan MS spectra were acquired in the Orbitrap
 analyzer. Scanning of the mass range was set to 350-1500 mass/load (m/z)
 at a resolution of 120,000 full width at half maximum. We used an
 automatic gain control (AGC) setting to 4.0 × 10<sup>5</sup>
 ions, maximum injection time to 50 ms, dynamic exclusion 1 at 90S and 10
 ppm mass tolerance. Subsequently, a top speed survey scan for 3 s was
 selected for subsequent decision tree-based Orbitrap CID or HCD
 fragmentation [16, 17]. The signal threshold for triggering an MS/MS event
 was set to 1.0 × 10<sup>4</sup> and the normalized collision
 energy was set to 35 and 30% for CID and HCD, respectively. The AGC of 3.0
 × 10<sup>4</sup> and isolation window of 1.6 m/z were set for
 both fragmentations. Additional parameters for CID included activation Q
 was set to 0.25 ms and injection time to 50 ms. For HCD, the first mass
 was set to 120 m/z and injection time to 100 ms. The settings for the
 decision tree were as follows: For HCD fragmentation charge states 2 or 3
 were scanned in a range of 650-1200 m/z, charge states 4 were scanned in a
 range of 900-1200 m/z, and charge states 5 were scanned in a range of
 950-1200 m/z; for CID fragmentation charge states 3 were scan in a range
 of 650-1200 m/z, charge state 4 were scan in a range of 300-900 m/z, and
 charge state 5 in scan range of 300-950 m/z. All data were acquired with
 Xcalibur 4.0.27.10 software (Thermo-Fisher Scientific).
 Mass spectra were analyzed with the Proteome Discoverer Program
 2.1 (PD, Thermo Fisher Scientific Inc.). The subsequent searches were run
 using Mascot server (version 2.4.1, Matrix Science, Boston, MA) and SQUEST
 HT [18]. The search with both engines was conducted against a translated
 database generated from <em>A. ludens</em> transcriptomic data
 [5]. The parameters of the analysis comprised: full-tryptic protease
 specificity, two missed cleavages allowed, static modifications covered
 carbamidomethylation of cysteine (+57.021 Da). Furthermore, dynamic
 modifications included methionine oxidation (+15.995 Da) and deamidation
 in asparagine/glutamine (+0.984 Da). For the MS2 (MS/MS or mass to the
 tandem) method, in which identification was performed at high resolution
 in the Orbitrap, precursor and fragment ion tolerances of ±10 ppm and ±
 0.2 Da were applied. The resulting peptide hits were filtered for maximum
 1% FDR using the Percolator algorithm [19].
 <strong>In silico protein annotation
 </strong> With the databases obtained from the
 Proteome Discoverer Program, a new database was created using a Biovenn
 diagram, considering only the proteins that were found in both replicates
 of reproductive tissues with two replicates (i.e., MAG, and reproductive
 tracts of unmated and mated females); for samples with only one replicate
 (i.e., bulb and testes), all proteins found were considered for further
 analysis. We submitted the FASTA files of proteins to a
 filtering process for the removal of duplicates. We searched for redundant
 proteins by first combining all protein sequences from all male and female
 tissues (n=3,096). We used CD-HIT-DUP (v0.0.1, under Galaxy platform https://usegalaxy.org/ using default parameters) to find duplicates. Based on our analysis of redundancy, we did not find redundant protein sequences.