Follicular cells Top-down proteomic
In mammals, the capacity of the female germ cell, the oocyte, to develop into embryo is acquired throughout meiotic maturation. Immature oocyte cannot be fertilized while mature oocyte is apt to accept spermatozoa and to develop an embryo. In a follicle, the oocyte is surrounded by mural granulosa cells (GC) and is physically and metabolically coupled with specialized granulosa cumulus cells (CC) which play an important role in oocyte maturation and fertilization. Factors expressing in GC and CC during maturation may reflect the oocyte quality, i.e. its capacity to be fertilized and assure early embryo development. However, the modifications of the content and the amount of peptide/proteins in the oocyte and the surrounding CC during oocyte maturation are mostly unknown and so there is not an accurate way of evaluating/monitoring how different in vitro maturation (IVM) protocols being in use in assisted reproduction technologies, can affect the process. In this context, Intact Cell MALDI-TOF Mass Spectrometry (ICM-MS) was applied to bovine follicular cells (bovine single oocytes, cumulus cells and granulosa cells) in order to characterize proteomic changes that occur in the follicle during female gamete development. In order to characterize finely endogenous molecular species observed on ICM-MS profiles and to identify markers of interest with their post-translational modifications, we carried out top-down proteomic on the different follicular cells from oocyte, oocyte-cumulus complexes, cumulus cells and granulosa cells protein extracts. Prior to top-down MS using a dual linear ion trap Fourier Transform Mass Spectrometer LTQ Orbitrap Velos, depending on the amount of available biological material, we employed three analytic strategies as a direct infusion, a mono-dimensional liquid chromatography (µLC-1D-MS/MS) and an off-line multi-dimensional liquid chromatography combining four fractionations (based on reverse phase or gel filtration LC) to µLC-MS/MS. Here, we deposited our dataset from µLC-1D-MS/MS (analyses of oocytes, oocyte-cumulus complexes, cumulus cells and granulosa cells protein extracts) and MDLC-MS/MS (analyses of granulosa cells protein extract).
Sample Processing Protocol
1) For Mono-dimensional Liquid Chromatography Top-Down HRMS : From granulosa cells, oocytes-cumulus complexes, cumulus cells and oocytes both at immature and mature state, proteins were extracted using Tris-Urea buffer (6 M Urea, 50 mM Tris-HCl pH 8.8 buffer containing protease inhibitor cocktail. Around 10-15 µg of protein extract were desalted using Zip Tip-C4. All fractions were analyzed by on-line micro-liquid chromatography tandem mass spectrometry (µLC-MS/MS) on the LTQ Orbitrap Velos mass spectrometer coupled to an Ultimate® 3000 RSLC Ultra High Pressure Liquid Chromatographer controlled by Chromeleon Software (version 6.8 SR11). Heigh microliters of each sample were loaded on a Dionex trap column (Monolithic PS-DVB PepSwift, 200 µm inner diameter x 5 mm long). Mobile phases consisted of (A) 0.1% formic acid, 97.9 % water, 2% acetonitrile (v/v/v) and (B) 0.1% formic acid, 15.9 % water, 84% acetonitrile (v/v/v). The separation was conducted using a Dionex column (Monolithic PS-DVB PepSwift, 200 µm inner diameter x 5 cm long). The flow rate was set to 1 µL/min. The linear gradient consisted of 4-95% B for 60 min. All experiments were performed using a LTQ Orbitrap Velos instrument operating in positive mode. Data were acquired using Xcalibur software v2.1. Standard mass spectrometric conditions for all analyses were spray voltage 1.1-1.4 kV, no sheath and auxiliary gas flow; heated capillary temperature, 275 °C; predictive automatic gain control enabled, and an S-lens RF level of 60%. Source fragmentation energy was set at 10 V. Full-MS in profile mode with subsequent data-dependent MS/MS analyses were acquired with a target resolution set to 100,000. In the scan range of m/z 400-2,000, the 10 most intense ions were selected and fragmented by HCD with normalized collision energy of 38% and wideband-activation enabled. Ion selection threshold was 500 counts for MS/MS with an isolation width = 3 m/z. The maximum allowed ion accumulation times were 200 ms for full scans (1 microscan) and 200 ms for HCD-MS/MS measurements (1 microscan). Target ion quantity for FT full MS was 1E6 and for MS/MS was 5E5. Dynamic exclusion was enabled with a repeat count of 2 and exclusion duration of 5,000 secondes. 2) For Multi-dimensional Liquid Chromatography Top-Down HRMS: In order to reduce sample complexity of granulosa cells protein extract, the proteins were subjected to pre-fractionations through reversed phase (RP) and gel filtration (GF) chromatographic separations on an UltiMate 3000 RSLC system controlled by Chromeleon v6.80 SR13 software. Additionally, one RP condition was combine to an ultracentrifugation process to enriched small proteoforms. For each condition, one mg of the peptides/proteins were injected and separated. A First approach (RP1) was based on RP HPLC using an XBridge BEH C18 column (250 × 4.6 mm i.d., particule size 5 μm; Waters). Mobile phases were (A) 0.1% (v/v) trifluoroacetic acid (TFA) in water, and (B) 0.1% (v/v) TFA in acetonitrile. The gradient elution was carried out at a flow rate of 1 mL/min with a linear gradient 10-60% B for 45 min. After vacuum-drying, all fractions were desalted using Zip Tip-C4. Another RP separation (RP2) was performed using a linear gradient: 2-60% for 43 min. Buffer A consisted of water with 0.1% TFA and buffer B consisted of 95% acetonitrile with 0.1% TFA. A third separation (RP3) consisted to combine centrifugal ultracentrifugation using Amicon® Ultra 0.5 mL centrifugal filters with a cut-off of 50 kDa (Millipore) to a RP separation. All the centrifugation steps were performed at a centrifugal force of 14,000 g for 15 min at 20°C. First, the centrifugal filter was rinsed with 0.1N NaOH followed by two rinses with water. Sample (2 mg) was diluted up to 500µL 10% (v/v) acetonitrile/5% (v/v) FA and loaded into the 50K filter device. After centrifugation, the filtrate was collected and 400 µL 10% acetonitrile/5% FA were added to the filter device. This resuspension and centrifugation process was repeated nine more times. Each time, filtrate was collected. At the end, all filtrates were pooled together and dried by vacuum-drying. The sample was dissolved in buffer A (0.1% TFA in water) and loaded onto the C18 RP-HPLC column for a RP separation under the same conditions as RP2. A fourth separation consisted to separate proteoforms by molecular weight using a Superdex 75 10/300 GL gel filtration (GF) column (GE Healthcare Europe GmbH) in 100 mM ammonium bicarbonate buffer. After vacuum-drying, all fractions were desalted using ZipTip C4. For RP1, RP2, RP3 and GF liquid chromatography processes, all fractions were analyzed by µLC-MS/MS analysis (as described in LC1D-MS/MS section at the exception of the use of longer analytic column (Monolithic PS-DVB PepSwift, 200 µm inner diameter x 25 cm long)).
Data Processing Protocol
For automated data acquired by µLC-MS/MS, raw files were automatically processed inside ProSightHT of the ProSight PC software v 3.0 SP1 (Thermo Fisher, San Jose). All data files (*.raw) were processed to group MS/MS data from different precursors of the same protein into one experiment for simultaneous analysis using the cRAWler application. Molecular weights of precursor and product ions were determined using the THRASH algorithm. All fragmentation data were filtered using the following parameters: signal/noise= 3/1, minimum fragment intensity at 100, retaining only the top 5 most intense neutral fragment masses within a 100 Da window below 2,000 Da. From PUF files, automated searches were performed using the “Biomarker and Absolute mass” search options against the database made via shotgun annotation from the UniprotKB Swiss-Prot Bos taurus release 2015_06 (bos_taurus_2015_06_top_down_simple.pwf downloaded from ftp://prosightpc.northwestern.edu/). For the biomarker searches, an iterative search tree was designed to begin with high mass accuracy (50 and 10 ppm at the intact and fragment ion level, respectively) for monoisotopic precursors. If the top result matched with a p-score of ≤1 × 10−6 the search engine accepted this result as valid. A second search was performed for invalid results using larger intact mass tolerances (average precursors with 3 Da mass tolerance). For all searches, all post-translation modifications were considered. First hits were automatically considered positively identified with a minimum of 5 matching fragment ions with E-value ≤1 × 10−6 for monoisotopic precursors and E-value ≤1 × 10−8 for average precursors. Then, all the .puf files were additionally searched in absolute mass mode against the dataset with 1,000 Da average precursor window and 10 ppm fragment tolerance. For identification of endogenous peptidoforms and proteoforms, the false discovery rate (FDR) was not estimated using decoy database in the current study; however, the conservative threshold for intact protein identifications by top-down using the Bonferroni-corrected Poisson model was significantly exceeded (minimum E-value at 9.9 E-6). Furthermore, according to recent top-down proteomic study showing that the E-value higher than 1 E-4 corresponded to 1% FDR threshold, we validated all the peptidoforms/proteoforms with E value < 1E-8 and > 1E- 5 only after being manually controlled using Sequence Gazer (sequence with at least 4 consecutive b or y fragment ions).
Labas Valerie, INRA-Nouzilly-PRC
Valérie Labas, Plate-forme d'Analyse Intégrative des Biomolécules et de Phénomique des Animaux d'Intérêt Bio-agronomique (PAIB2) UMR INRA 85 - CNRS 7247 - UFR - IFCE Physiologie de la Reproduction et des Comportements INRA Centre Val de Loire (Tours) 37380 NOUZILLY ( lab head )
Labas V, Teixeira-Gomes AP, Bouguereau L, Gargaros A, Spina L, Marestaing A, Uzbekova S. Intact cell MALDI-TOF mass spectrometry on single bovine oocyte and follicular cells combined with top-down proteomics: A novel approach to characterise markers of oocyte maturation. J Proteomics. 2017 Apr 3. pii: S1874-3919(17)30118-5 PubMed: 28385661