Label-Free Proteomics Uncovers Energy Metabolism and Focal

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Label-free Proteomics Uncovers Energy Metabolism and Focal Adhesion Regulations Responsive for Endometrium Receptivity Qian Chen, Aijun Zhang, Feng Yu, Jing Gao, Yue Liu, Chengli Yu, Hu Zhou, and Chen Xu J. Proteome Res., Just Accepted Manuscript • Publication Date (Web): 02 Mar 2015 Downloaded from http://pubs.acs.org on March 3, 2015

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Journal of Proteome Research

Label-free Proteomics Uncovers Energy Metabolism and Focal Adhesion Regulations Responsive for Endometrium Receptivity

Qian Chen

1,2,#

, Aijun Zhang

2,#

, Feng Yu3, Jing Gao3, Yue Liu1, Chengli Yu3, Hu

Zhou3,4,* and Chen Xu1,*

1

Department of Human Anatomy, Histology and Embryology, Shanghai Jiao Tong

University School of Medicine, Shanghai Key Laboratory for Reproductive Medicine, Shanghai 200025, China,

2

Center of Reproductive Medicine, Ruijin Hospital,

Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China, 3CAS Key Laboratory for Receptor Research, Shanghai Institute of Materia Medica, Shanghai 201203, China, 4E-institute of Shanghai Municipal Education Committee, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China

#

These authors have contributed equally to this work

*

To whom correspondence may be addressed: Chen Xu, Department of Human Anatomy, Histology and Embryology, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory for Reproductive Medicine, Shanghai, China，200025， Email: [email protected] Hu Zhou, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Zhangjiang Hi-Tech Park, Shanghai, China, 201203, Email: [email protected]

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ABSTRACT The menstrual cycle of the female uterus leads to periodic changes of the endometrium. These changes are important for developing the endometrial receptivity and for achieving competency of embryo implantation. However, the molecular events underlying the endometrial receptivity process remain poorly understood. Here, we applied a LC-MS based label-free quantitative proteomic approach to compare the endometrial tissues in the mid-secretory (receptive) phase with the endometrial tissues in the proliferative phase from age-matched woman (n=6/group). The proteomes of endometrial tissues were extracted using an SDS-based detergent, digested by the filter aided sample preparation (FASP) procedures, and subsequently analyzed by nano LC-MS/MS (Orbitrap XL) with a 4-h gradient. Reliable protein expression profiles were reproducibly obtained from the endometrial tissues in the receptive and proliferative phases. A total of 2138 protein groups were quantified under highly stringent criteria with a false discovery rate of 100s) were found to be changed with statistical significance and only a few genes, including osteopontin, olfactomedin, apolipoprotein D, and dickkopf/DKK1, were found to be consistently differentially expressed in all of the studies (15). These results demonstrated the developmental complexity of the endometrium. Considering that the microarray results cannot fully represent the protein changes due to the post-transcriptional and post-translational regulations, proteomic approaches have also been used to study those differentially expressed proteins in the endometrial tissues of different phases (16-25). Byrjalsen et al. (16) demonstrated the first analysis by two-dimensional gel electrophoresis (2-DE) of the human endometrial tissue in the proliferative, interval, and secretory phases, and a total of 1095 and 488 [35S]-labelled protein spots were emerged by the iso-electric focusing gels (pI 3.5-7) and non-equilibrium pH gradient electrophoresis (pI 6.5-11), respectively. However, no proteins were identified in this study. Parmar et al. (21) applied 2-DE and MALDI-TOF/TOF mass spectrometry to identify the proteins that are differentially expressed in the mid-secretory (receptive) phase of the endometrium compared to the proliferative phase of the endometrium. More than 500 protein spots were displayed in the 2-DE map of the two phases of the endometrial


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tissues by densitometry analysis which included 104 spots up-regulated and 57 spots down-regulated in the receptive phase. Eight spots with more than a 2-fold change between the two phases were identified by MALDI-TOF-TOF, resulting in the identification of four down-regulated proteins in the receptive phase (i.e. calreticulin, fibrinogen, adenylate kinase isoenzyme 5, and transferring) and four up-regulated proteins in the receptive phase, including annexin V, peroxidoxin 6, a1-antitrypsin (AAT), and creatine kinase (21). Dominguez et al. (18) used two-dimensional differential in-gel electrophoresis (2D-DIGE) and MALDI-TOF/TOF to analyze the proteome of the human receptive versus pre-receptive endometrium. A total of 2500 spots per gel were detected, but only 32 unique proteins were identified with significant changes between the pre-receptive and receptive endometrium. Chen et al (17) employed 2D differential in-gel electrophoresis (DIGE) to analyze the endometrial proteome between the proliferative and secretory phases, and obtained 196 differentially expressed spots from a total of 1017 spots. Among them, 76 proteins, including Rho-GDIα, CLIC1 and PGRMC1, were identified by mass spectrometry. Their following pathway analysis revealed that Jnk and EGF signaling pathways were significantly enriched for regulation of endometrial protein expression in the midsecretory phase (17). Rai et al. (22) reported that 200 spots were detected and 194 protein spots were identified by 2-DE-MS from the proteome of human endometrium between the proliferative and secretory phases. Hannan et al (19) employed 2D-differential in gel electrophoresis (2D-DiGE) to analyze the endometrial secretomes from receptive and nonreceptive phases in fertile and infertile women, and they found several soluble proteins that may be essential to endometrial receptivity and can be used as potential diagnostic markers and treatment targets. Li et al. (20) analyzed the endometrial tissue samples from the pre-receptive and receptive phases after a luteinizing hormone surge, and 2,555±98 protein spots and 167 differentially expressed proteins spots were found to be present in the overlaid 2-DE map while finally 31 differentially expressed proteins were identified by MALDI-TOF. In the above-mentioned 2-DE based studies, reproducible pattern of protein spots that represents the global endometrium proteome could be obtained. However,



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minority of the proteins on the gel could be identified and therefore hinders the global systematic analysis including those low abundant proteins and modestly regulated proteins. Also it is tedious and time-consuming process to individually extract, digest, and analyze each spot. In this regard, DeSouza et al. (26) employed isotope-coded affinity tags (ICAT) and nano-LC-MS/MS to analyze the proteome of the human proliferative and secretory phases of the endometrium. They obtained a list of 119 proteins by a manual inspection of MS/MS data and the additional searches using MASCOT. DeSouza et al (27) used mTRAQ-labeling and multiple reaction monitoring (MRM) technology to quantify pyruvate kinase-M1/M2 in nonmalignant endometrial tissue of the proliferative phase and an endometrial tumor sample. Vehmas et al (28) analyzed the tissues from patient endometrium and ovarian endometrioma using a semi-targeted approach that combines accurate inclusion mass screening (AIMS), inclusion lists, and LC−MS label-free quantitation, and they found that TGFβ1 regulatory network was activated in ovarian endometriosis development. Meng et al. (29) employed the iTRAQ approach to analyze the endometrium samples from 12 women. Out of these endometria samples, four had natural cycles, for had controlled ovulation stimulation with a GnRH agonist long protocol, and another four samples were subjected to a GnRH antagonist protocol. As a result, 1938 proteins were detected from all three groups. 24 proteins of these proteins were differentially expressed between the natural cycle

and the GnRH agonist treatment group;

whereas, 39 proteins were differentially expressed between the natural cycle (N) and the GnRH antagonist treatment group (29). Nevertheless, extensive and reliable expression profiling of the endometrial proteome remains a prominent challenge due to the high percentage of integral membrane and membrane-associated proteins in the endometrial tissues. Label-free proteomic analysis is a newly emerged, efficient, powerful and cost-effective (compared to iTRAQ or ICAT workflow mentioned above) approach for comparing multiple clinic samples from different individuals and till now has not been applied in the proteomic analysis of receptive and proliferative endometrial tissues. In order to


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achieve reliable and relative quantification, reproducible sample preparation and LC-MS/MS analyses are crucial for large-scale sample cohort analysis. Considering the technical insights and the pressing need for the further understanding of the endometrial remodeling process (despite of the previous proteomic studies), in the present study, we performed a reproducible, well-controlled, label-free quantitative proteomic analysis of the endometrial tissues in the receptive and proliferative phases (n=6 women per group). Specifically, to universally extract the proteins from endometrial tissues, an SDS-containing lysis buffer was used to efficiently extract proteins from the endometrial tissues, and then the samples underwent a FASP procedure (30). Secondly, a one-dimensional 4-h nano-LC-MS analysis was used to ensure an extensive and also robust comparative analysis for the 12 individual endometrial tissue samples. A total of 2138 unique proteins were quantified, of which, 317 proteins were significantly changed between the receptive and proliferative phases. The altered proteins were subjected to pathway and direct protein-protein interaction analysis, and importantly, the evidenced and thus selected protein events of interest were carefully validated by Western blot and indirect immunofluorescence analysis. These results provide novel evidence that molecular changes of the endometrial tissues in the receptive and proliferative phases may be associated with the physiological and biological mechanisms that underlie endometrial receptivity.

MATERIALS AND METHODS Endometrial Tissue Biopsy Sampling Ethical approval was obtained from the Institutional Ethics Committee of the Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University for all tissue collections. Written informed consent was obtained from all subjects. Twelve women between ages 26 and 32 years, who were attending the Reproductive Medical Center of Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University for IVF treatment, were recruited for this study. All of the women had regular menstrual cycles and normal uteruses. No significant intrauterine or ovarian abnormalities were



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detected by transvaginal ultrasonography. Patients with uterine abnormalities such as leiomyomas and endometrial polyps were excluded from the study. Only women with tubal factor or male factor infertility, free of steroidal hormones for two or more months prior to this study, and who agreed to use condoms for contraception during the study were recruited for endometrial biopsies. Furthermore, the analysis was restricted to women who had given birth to live babies after subsequent IVF treatments following the study. After the baseline endocrine investigation on day 3 of the menstrual cycle, serial ultrasonography was performed to document ovulation. From days 7-9 of the menstrual cycle, ultrasonography was done every other day until the diameter of the dominant follicle was ＞15 mm. At this stage, ultrasonography was done daily until there was evidence of follicular rupture. Proliferative phase samples were collected on days 7-9 of the menstrual cycle. In these women, serial ultrasonography was continued until the follicle ruptured. The samples were also checked by histology to ensure the presence of proliferative and secretory endometrium. Endometrial tissue samples were obtained using a single use S type endometrial biopsy tube (TY-C3.1/30-1S, TianYi，Zhejiang, China) from six women in their mid-secretory phase (on day 7 after ovulation) and from six women during their proliferative phase (on day 7-9 of the menstrual cycle). The samples were washed immediately in normal saline to completely remove the blood inside and divided into two parts: one part was placed in 10% buffered formalin for paraffin embedding, and the other part was placed in liquid nitrogen. The total duration from endometrial biopsy removal to sample freezing was controlled to be in 5 minutes, and this short cold ischemia time cannot affect the global proteome quantitation (31).

Sample Preparation Human endometrial tissues were lysed as described (32). Briefly, each tissue was added to SDS-lysis buffer (2% SDS, 0.1 M DTT, 0.1 M Tris-HCl, pH=7.6), and the ratio of buffer to tissue was 10:1. After homogenization for 3 min, the mixture was incubated in boiling water for another 3 min. Then, the crude extract was sonicated


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for 1 min and centrifuged at 16,000 g for 30 min at 20 °C. The protein concentration was detected by measurements of tryptophan fluorescence as previously described (33). Briefly, 1 µL of sample or tryptophan standard (100 ng/µL) was added into 3 mL of 8 M urea buffer (8 M urea, 20 mM Tris-HCl, pH=7.6). Fluorescence was excited at 295 nm and measured at 350 nm. The slits were set at 10 nm.

Protein Trypsin Digestion Using FASP Method 100 µg of sample was digested by the FASP procedure as previously described (32). Each sample was transferred to a 10 K filter (Pall Corporation) and centrifuged at 14,000 g for 40 min at 20 °C. Then, 200 µL of urea buffer (8 M urea, 0.1M Tris-HCl, pH=8.5) was added to the sample and centrifuged at 14,000 g for another 40 min. This step was repeated one more time. The concentrate was then mixed with 100 µL of 50 mM IAA in urea buffer and incubated for an additional 40 min at room temperature in darkness. After that, IAA was removed by centrifugation at 14,000 g for 40 min. Next, the sample was diluted with 200 µL of urea buffer and centrifuged two more times. Then, 200 µL of 50 mM NH4HCO3 was added and the sample was centrifuged at 14,000 g for 40 min. This step was repeated twice. Finally, 100 µL of 50 mM NH4HCO3 and trypsin (1:50, enzyme to protein) were added to the sample which was then incubated at 37 °C for 16 h. The tryptic peptide mixtures were collected for further analysis. LC-MS/MS Analysis The reverse phase high-performance liquid chromatography (RP-HPLC) separation was achieved on the Suveyor HPLC system (Thermo Fisher Scientific) using a self-packed column (75µm × 120mm; 3 µm ReproSil-Pur C18 beads, 120 Å , Dr.Maisch GmbH, Ammerbuch, Germany) at a flow rate of 250 nL/min. The mobile phase A of RP-HPLC was 0.1% formic acid in water, and B was 0.1% formic acid in acetonitrile. The peptides were eluted using a gradient (2–80% mobile phase B) over a 4 h period into a nano-ESI linear ion trap (LTQ)-Orbitrap XL mass spectrometer (Thermo Fisher Scientific). The mass spectrometer was operated in data-dependent mode with each full MS scan followed by MS/MS for the 10 most intense ions with



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the parameters: ≥ +2 precursor ion charge, 2 Da precursor ion isolation window and 35 normalized collision energy of LTQ. The following Dynamic Exclusion™ settings were also used: repeat counts, 1; repeat duration, 120 s; exclusion duration, 180 s. The full mass was scanned in the Orbitrap analyzer with R = 60,000 (defined at m/z 400), and the subsequent MS/MS analyses were performed in the LTQ analyzer (resolution: FWHM=0.5). The MS data were analyzed using the software MaxQuant (34-35) (http://maxquant.org/, version 1.3.0.5). Carbamidomethyl (C) was set as a fixed modification, and oxidation (M, +15.99492 Da) was set as a variable modification. Proteins were identified by searching MS and MS/MS data of peptides against a decoy version of the International Protein Index (IPI) human database (version 3.87, 91464 protein sequences; European Bioinformatics Institute). Trypsin/P was selected as the digestive enzyme with two potential missed cleavages. The false discovery rate (FDR) for peptides and protein groups was rigorously controlled to be

Label-Free Proteomics Uncovers Energy Metabolism and Focal

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