| Metadata |
| datasetIdentifier | PASS00436 |
| datasetType | MSMS |
| submitter | Matthew Champion <mchampio@nd.edu> |
| submitter_organization | University of Notre Dame |
| lab_head_full_name | Norman J Dovichi |
| lab_head_email | ndovichi@nd.edu |
| lab_head_organization | PI |
| lab_head_country | United States |
| datasetTag | Sun_Xenopus_iTRAQ |
| datasetTitle | Quantitative proteomics of Xenopus laevis embryos: expression kinetics of nearly 4000 proteins during early development, using multidimensional chromatography with iTRAQ isobaric quantification |
| publicReleaseDate | 2014-02-28 00:00:00 |
| finalizedDate | |
| summary | Multiple embryo stages (1,8,13,22) on single and 4-pooled embryos from X. laevis were digested, labeled with ITRAQ-8 plex and identified and quantified using multiplecation exchange fractions. |
| contributors | Liangliang Sun*, Michelle M. Bertke, Matthew M. Champion**, Guijie Zhu, Paul W. Huber, Norman J. Dovichi+
* Primary Author
** Submitter
+ Corresponding author |
| publication | Quantitative proteomics of Xenopus laevis embryos: expression kinetics of nearly 4000 proteins during early development
Liangliang Sun, Michelle M. Bertke, Matthew M. Champion, Guijie Zhu, Paul W. Huber, Norman J. Dovichi*
Scientific Reports, (In Revision) |
| growth | Xenopus laevis embryo culture and collection. All animal procedures were performed according to protocols approved by the University of Notre Dame Institutional Animal Care and Use Committee. Female Xenopus laevis were induced to lay eggs by injection with 600 units of human chorionic (C1063, Sigma-Aldrich, St. Louis, MO) 12-15 hours prior to spawning. Testes were isolated from anesthetized males at the time of spawning. Eggs and minced testis were combined in 1/3 MMR (Marc’s Modified Ringers) for fertilization and embryos were maintained at room temperature and throughout development. Embryos were collected at different stages based on the information from60-61. Eight eggs were collected separately into eight different Eppendorf tubes at stage 1. Then, six embryos were collected at stages 5, 8, 11, 13, and 22. A single embryo was collected into one Eppendorf tube. |
| treatment | Xenopus laevis embryo culture and collection. All animal procedures were performed according to protocols approved by the University of Notre Dame Institutional Animal Care and Use Committee. Female Xenopus laevis were induced to lay eggs by injection with 600 units of human chorionic (C1063, Sigma-Aldrich, St. Louis, MO) 12-15 hours prior to spawning. Testes were isolated from anesthetized males at the time of spawning. Eggs and minced testis were combined in 1/3 MMR (Marc’s Modified Ringers) for fertilization and embryos were maintained at room temperature and throughout development. Embryos were collected at different stages based on the information from60-61. Eight eggs were collected separately into eight different Eppendorf tubes at stage 1. Then, six embryos were collected at stages 5, 8, 11, 13, and 22. A single embryo was collected into one Eppendorf tube.
The digests were acidified with formic acid to terminate the tryptic digestion, followed by peptide desalting with C18 spin columns (Pierce Biotechnology, Rockford, IL) for E1 and E2 samples, and with Sep-Pak C18 1 cc Vac Cartridge (Waters Corporation, Milford, MA) for E3 samples. After lyophilization, the peptides were labeled by ‘isobaric tags for relative and absolute quantitation’ (iTRAQ) 8-plex reagents according to the manufacturer’s protocols (AB Sciex, Foster City)22 with the following minor modifications. The lyophilized digests for E1 and E2 were dissolved in 12.5 µL of dissolution buffer, and the digests for E3 were dissolved in 35 µL of dissolution buffer. After addition of 50 µL of isopropanol to each iTRAQ reagent vial, 25 µL of iTRAQ reagent was added to the digest for E1 and E2, and an entire vial of iTRAQ reagent was used to label the digests for E3. After labeling at room temperature for 2 hours, 35 µL of 100 mM Tris-HCl buffer (pH 8.0) was added to the samples for E1 and E2 and incubated at room temperature for 40 min to block the residual iTRAQ reagents. For E3 samples, 100 µL of 100 mM Tris-HCl buffer (pH 8.0) was used. Then, the labeled samples in each experiment were mixed, and three tubes of labeled digests (E1, E2 and E3) were obtained. After lyophilization, the samples were dissolved in 500 µL (E1 and E2) or 800 µL (E3) of 2% ACN and 0.1%FA solution, followed by desalting with Sep-Pak C18 1 cc Vac Cartridge (Waters). The digests were lyophilized again, and then redissolved in 250 µL of 0.1% FA, followed by strong cation exchange (SCX) liquid chromatography fractionation. |
| extraction | Embryo lysis and protein sample preparation. Each collected embryo was suspended in 50 µL of mammalian cell-PE LBTM buffer containing complete protease inhibitor. After shaking for 30 s at room temperature, the tubes were sonicated for 3 min on ice with a Branson Sonifier 250 (VWR Scientific, Batavia, IL) to lyse the embryos completely. The lysates were then kept on ice for half an hour. The tubes were next centrifuged at 12,000 g for 10 min. Finally, the supernatants were collected into fresh Eppendorf tubes and stored at -80 oC before use.
The embryo lysate supernatants were precipitated with 300 µL of cold acetone at -20 oC for 6 h. After centrifugation, the supernatants were removed, and another 300 µL of cold acetone was added to each tube to wash the pellets again. After further centrifugation, the supernatants were removed and protein pellets were dried at room temperature. |
| separation | The embryo lysate supernatants were precipitated with 300 µL of cold acetone at -20 oC for 6 h. After centrifugation, the supernatants were removed, and another 300 µL of cold acetone was added to each tube to wash the pellets again. After further centrifugation, the supernatants were removed and protein pellets were dried at room temperature.
SCX fractionation. The labeled samples from three experiments (E1, E2 and E3) were fractionated by SCX liquid chromatography using a Waters Alliance HPLC system (Waters, Milford, MA, USA) at a flow rate of 0.25 mL/min. About 150 µL of the labeled peptide samples was loaded onto an SCX guard column (4.6 mm i.d. ×12.5 mm length, Agilent Technologies, Wilmington, DE, USA), and then separated with a Zorbax 300-SCX column (2.1 mm i.d. × 50 mm length, 5 µm particles, Agilent Technologies). The mobile phase gradient was generated using buffer A (10 mM KH2PO4, 20% ACN, pH 2.85) and buffer B (1 M KCl in A, pH 2.85). The samples in 0.1% FA were loaded, followed by 10 min washing with 100% A to remove excess iTRAQ reagent. Then, the peptides were separated by a 25 min linear gradient from 100% A to 100% B. Finally, the column was washed by 100% B for 5 min, followed by column equilibration with 100% A. Fractions were collected from 12 min to 42 min as follows. Eluate from 12 min to 18 min was collected to one fraction and from 36 min to 42 min as one fraction. From 18 min to 36 min, the eluate was collected as 1 min/fraction. In total, 20 fractions were collected from each sample. |
| digestion | The pellet in each tube was dissolved in 30 µL of 8 M urea, 100 mM NH4HCO3 (pH 8.0) buffer via vortex with sonication. After centrifugation, an 8 µL aliquot of protein solution was taken from each tube and further diluted to 24 µL with 100 mM NH4HCO3 (pH 8.0), followed by protein concentration measurement using the bicinchoninic acid (BCA) method62. For experiment I (E1), two embryos from stage 1, 5, 8, and 11 were used and each embryo lysate was further prepared individually. For experiment II (E2), two embryos from stage 1, 5, 13, and 22 were used and each embryo lysate was also further prepared individually. For experiment III (E3), the mixtures of four embryos from stage 1, 8, 13, and 22 were used for further preparation. All the samples were denatured at 37 oC for 1 h, followed by protein reduction in 20 mM DTT at 56 oC for 1 h and alkylation in 50 mM IAA at room temperature for 30 min in dark. The treated samples were further diluted four times with 100 mM NH4HCO3 (pH 8.0) to reduce the urea concentration to about 2 M, followed by trypsin digestion at 37 oC overnight with trypsin/protein mass ratio as 1/25. |
| acquisition | UPLC-ESI-MS/MS analysis. A nanoACQUITY UltraPerformance LC® (UPLC®) system (Waters, Milford, MA, USA) was used for peptide separation. Buffer A (0.1% FA in water) and buffer B (0.1% FA in ACN) were used as mobile phases for gradient separation. Peptides were automatically loaded onto a commercial C18 reversed phase column (Waters, 100 µm×100 mm, 1.7 µm particle, BEH130C18, column temperature 40 ℃) with 2% buffer B for 10 min at a flow rate of 1 µL/min, followed by 3-step gradient separation, 2 min from 2% to 8%, 114 min to 28% B, 3 min to 85% B, and maintained at 85% B for 13 min. The column was equilibrated for 12 min with 2% B before analysis of the next sample. The eluted peptides from the C18 column were pumped through a capillary tip for electrospray, and analyzed by a Q-Exactive mass spectrometer (Thermo Fisher Scientific). For each sample, 2 µL of peptides were used for analysis.
The electrospray voltage was 1.6 kV, and the ion transfer tube temperature was 280 ˚C. The S-Lens RF level was 50.00. The data acquisition was programmed in data dependent acquisition (DDA) mode. A top 12 method was used. Full MS scans were acquired in Orbitrap mass analyzer over m/z 380-1800 range with resolution of 70,000 (m/z 200) and the number of microscans set to 1. The target value was 1.00E+06, and maximum injection time was 250 ms. For MS/MS scans, the twelve most intense peaks with charge state ≥ 2 were sequentially isolated and further fragmented in the higher-energy-collisional-dissociation (HCD) cell following one full MS scan. To reduce the interference of peptide co-fragmentation to the iTRAQ quantitation, the isolation window was set as 1.0 m/z. The normalized collision energy was 33%, and tandem mass spectra were acquired in the Orbitrap mass analyzer with resolution 35,000 (m/z 200). The fixed first mass was m/z 100.0. The target value was 1.00E+06 and maximum injection time was 120 ms. The number of microscans was 1 and the ion selection threshold was 5.0E+04 counts. Peptide match and exclude isotopes were turned on. Dynamic exclusion was set as 30 s. |
| informatics | Data analysis. The .raw files were converted to Mascot generic format (.mgf) files via RAW2MSM software33 with default settings for deep proteome analysis, and via Proteome Discoverer 1.3 (Thermo Fisher Scientific) with default settings for protein quantitation analysis. Protein PilotTM 4.5 (AB Sciex, Foster City, CA, USA) was used for deep proteome analysis and protein quantitation analysis with .mgf files as input. ParagonTM algorithm (v. 4.5.0.0, 1654)63 integrated in the Protein PilotTM 4.5 was used for database searching. The Xenopus laevis database (12/28/2012 version) downloaded from the Xenbase website (ftp://ftp.xenbase.org/pub/Genomics/Sequences/). The Xenopus database was then combined with common contaminants and used for database searching. The parameters for database searching were as follows: iTRAQ 8plex (peptide labeled) was set as sample type. Iodoacetamide was set as the cysteine alkylation, trypsin as the digestion enzyme, Orbitrap MS (1-3 ppm) and Orbitrap MS/MS as instrument and urea denaturation as special factors. In addition, search effort was set as “thorough” for deep proteome analysis, and “rapid” for protein quantitation. The database searching for the reversed database was also performed in order to evaluate FDRs at the peptide and protein levels64-65.
For deep proteome and protein quantitation analysis, the peptide confidence was filtered to produce a peptide level global FDR of less than 1%. On protein group level, the protein unused score was used to filter the protein identification to produce a protein level FDR less than 1%. For protein quantitation, only ratios from the spectra that were unique to each protein were used for calculation of protein ratio, and only “Auto” peptides were used for protein quantitation. Bias correction was applied for protein quantitation results, which determines the median average protein ratio and corrects it to unity, and then applies this factor to all quantitation results. Proteins with iTRAQ ratio higher than 20 or lower than 0.05 were not considered as quantified, and only proteins with reasonable ratios across all channels were recognized as quantified ones. We obtained the final protein quantitation information based on normalization to the mean of channels 113 and 114 (biological replicate of stage 1) for E1 and E2, and the mean of iTRAQ protein ratios of biological replicates for each embryonic stage was used for further data analysis. We calculated the FDR for our quantitative data using a target-decoy approach by comparing ratios on duplicate iTRAQ channels.
DAVID Bioinformatics Resources 6.735 was used to generate the gene symbols of the Xenopus laevis proteome, biological process, molecular function and cellular component information.
Open source software, GProX66 used to visualize protein quantitation data including histograms and clustering analysis. The log2 ratios were used for analysis. For histograms analysis, default settings were used. For clustering analysis, the number of clusters was set to 6, and fixed regulation threshold (upper limit as 0.26 and lower limit as -0.32, corresponding to the original ratios of about 1.2 and 0.8) was used. The minimal membership for plot was set as 0.5. Other parameters were default settings. |
| instruments | Thermo LTQ Orbitrap |
| species | Xenopus laevis |
| massModifications | variable: K,Y,H,NT+304.2054
variable: M+16.0001
static: C+57.0214 |
