Proteomic Characterization of Midproliferative and Midsecretory Human Endometrium Jenny I-C. Chen,* Natalie J. Hannan, Yunxian Mak, Peter K. Nicholls, Jin Zhang, Adam Rainczuk, Peter G. Stanton, David M. Robertson, Lois A. Salamonsen, and Andrew N. Stephens Prince Henry’s Institute of Medical Research, P.O. Box 5152, Clayton, Victoria 3168, Australia Received November 26, 2008
This study aimed to identify proteins differentially expressed in the human endometrium between the proliferative and secretory phases of normal menstrual cycles by 2D differential in-gel electrophoresis (DIGE). A total of 196 out of 1017 spots were differentially expressed (p < 0.05). Mass spectrometry identified 76 proteins representing 41 different gene products. Immunohistochemistry confirmed the observed changes in 3 representative proteins (Rho-GDIR, CLIC1, PGRMC1). Biological pathway analysis identified the Jnk and EGF signaling pathways as key regulators of protein expression in the midsecretory phase of endometrial proteome. Keywords: Endometrium • 2D PAGE • two-dimensional differential in-gel electrophoresis (2D DIGE) • proteome • proliferative and secretory phases of the menstrual cycle • mass spectrometry • Rho-GDIR • CLIC1 • PGRMC1
Introduction The human endometrium is a highly dynamic tissue that undergoes cyclical remodeling with every menstrual cycle during the reproductive years.1 Almost the entire functional layer of the endometrium is shed during menstruation, with subsequent regeneration of the tissue from the underlying basal layer. The menstrual cycle is broadly divided into three phases, the menstrual phase, the proliferative phase, which occurs prior to ovulation when cellular proliferation is dominant, and the secretory phase following ovulation, when differentiation of all the cell types occurs. The purpose of this remodeling is to provide an environment that is conducive to implantation of the conceptus, but only at a time when the conceptus is appropriately developed. Interestingly, developmental plasticity which is generally lost in adult tissues is retained in the endometrium since this tissue constantly renews during adult life. Disturbances to this normal endometrial remodeling underpin many of the major problems in women’s health. For example, infertility (which affects 1 of every 10 couples) can result from inadequate or out-of-phase differentiation of the endometrium during the secretory phase, while development of endometrial cancer occurs when normal control of endometrial proliferation is lost. The genomics of endometrial cyclicity has been addressed in a wide range of studies2,3 and compelling evidence now exists regarding changes in gene expression profiles that define the different phases of the human menstrual cycle.4-6 Such studies have defined the potential of gene expression profiles for developing molecular diagnoses of * To whom correspondence should be addressed. Prince Henry’s Institute; P.O. Box 5152, Clayton, Victoria 3168 Australia. Phone: +61 3 9594 7919. Fax: +61 3 9594 6125. E-mail:
[email protected].
2032 Journal of Proteome Research 2009, 8, 2032–2044 Published on Web 02/17/2009
normal versus abnormal endometrium and for identifying molecular targets for potential therapeutic purposes. However, genomic studies have well-recognized limitations: not all transcripts are translated and many proteins undergo considerable post-translational modifications. The one study to date comparing proliferative and secretory phase endometrium used isotope-coded affinity tags (ICAT) and identified 119 proteins in endometrium, only 5 of which showed consistent differential expression; only 2 of these were confirmed.7 The aims of the present study were to compare the protein profiles of midproliferative and midsecretory phase endometrium by differential in-gel electrophoresis (DIGE), to identify the differentially expressed proteins by mass spectrometry (MS), to determine the complementarity of the data obtained with published gene profiles and to validate some of the differentially produced proteins and their cellular location by immunohistochemistry. These studies will lay the groundwork for further studies that will define proteins (particularly their various isoforms) important for establishing endometrial receptivity and for development of endometrial cancers.
Materials and Methods Tissue Collection and Patient Details. Ethical approval was obtained from appropriate Institutional Ethics Committees for all tissue collections. Written informed consent was obtained prior to tissue collection from all subjects. Human endometrial biopsies were obtained by curettage during the midproliferative (days 8-10; n ) 10) and midsecretory (days 19-23; n ) 10) phases of the menstrual cycle from normal fertile women undergoing curettage following laparoscopic sterilization or assessment of tubal patency. Patients with uterine abnormalities such as leiomyomas, endometrial polyps, endometriosis, or those who had received steroid hormone therapy in the last 10.1021/pr801024g CCC: $40.75
2009 American Chemical Society
Cyclical Changes in Endometrial Proteome 6 months were excluded. Cycle stage was confirmed by histological dating, according to standard criteria.8 Endometrial biopsies were divided in two: one portion was fixed in 10% buffered formalin overnight (17 ( 1 h), then washed three times in Tris-buffered saline (TBS, pH 7.6) before routine histological processing to paraffin blocks. The remainder was placed into RNAlater solution (Ambion, Austin, TX) before snap-freezing and stored at -80 °C for subsequent protein extraction. Protein Extraction. Snap-frozen endometrial tissue samples from the midproliferative (n ) 4, age range 30-35, median age 32) and midsecretory (n ) 4, age range 31-36, median age 33) phases were washed three-times in 10 mM Tris/HCl (pH 7.4) and homogenized in 0.8 mL of reagent (40 mM Tris/HCl, 7 M urea, 2 M thiourea, 1% (w/v) C7BzO) using a Heidolph DIAX 600 homogenizer (Heidolph, Germany). Following centrifugation (16 000g × 5 min at 4 °C), the supernatant was transferred to a fresh tube and the pellet re-extracted as above. The two supernatants were combined and the proteins precipitated in 10 vol of acetone at room temperature for 90 min. Protein was recovered by centrifugation at 10 000g (Beckman JS7.5 rotor) for 20 min at room temperature. The protein pellet was airdried and then dissolved in CyDye labeling buffer at room temperature (30 mM Tris/HCl, pH 8.1, 7 M urea, 2 M thiourea, 1% (w/v) C7BzO). Protein concentration was determined using a modified Bradford method.9 The final protein concentration was adjusted to 5 mg/mL with Cydye labeling buffer, snapfrozen on dry ice and stored at -80 °C. DIGE Labeling of Proteins. Fluorescent labeling of proteins was performed using minimal CyDye labeling methodology as described by the manufacturer (GE Healthcare Bioscience, Uppsala, Sweden). In brief, labeling was carried out on ice, in the dark for 30 min using 200 pmol dye per 50 µg protein at a protein concentration of 5 µg/µL. The labeling reaction was quenched by the addition of 1/10 vol of 10 mM lysine. Proliferative or secretory phase protein samples were labeled using Cy3 or Cy5, respectively, while an internal standard (comprised of equal amounts of protein from each sample) was labeled with Cy2. On completion of labeling, the Cy3 and Cy5 labeled samples were combined pairwise and an aliquot of Cy2-labeled internal standard was added to each. Each sample was then precipitated in acetone as described above, and the dried protein pellet resuspended in 100 µL of the solubilization reagent (7 M urea, 2 M thiourea, 1% (w/v) C7BzO) prior to isoelectric focusing. Samples were snap-frozen on dry ice and stored at -80 °C until use, typically within 1 day of labeling. Two Dimensional Polyacrylamide Gel Electrophoresis (2D PAGE). Isoelectric focusing (IEF) was performed using 24 cm immobilized pH gradient strips (IPGs) covering the pH range of 4-7. Tracking dye, DTT to 100 mM and ampholytes to 0.5% (v/v) were added and each sample was loaded by passive rehydration for 6 h at room temperature. IPG strips were focused overnight using an IPGphor instrument (GE Healthcare, BUCKS, U.K.). Focusing was carried out according to the following profile: current limited to 60 µA per strip, 100 V for 90 min, 300 V for 90 min, 500 V for 3 h, gradient to 1000 V for 4 h, gradient to 8000 V for 3 h, and then constant 8000 V overnight until reaching between 60 and 80 000 Vh. Following focusing, the strips were frozen at -80 °C overnight. Prior to electrophoresis, the IPG strips were thawed and incubated in 10 mL of equilibration buffer (50 mM Tris/HCl, pH 8.8, 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS, 0.01% (w/v) bromophenol blue) for 20 min on a shaking platform at 80 rpm. Second-
research articles dimension PAGE was performed on 8-16% gradient polyacrylamide gels (24 × 24 cm) overnight in a BioRad Dodeca cell (Richmond CA) at 50 V as described by the manufacturer. The run was terminated when the dye front had reached the bottom of the gel. Image Capture and Analysis. 2D gels were scanned using a Fuji FLA5100 laser scanner (FujiFilm, Tokyo, Japan) at excitation wavelengths of 473, 535, and 635 nm, and with specialty dual wavelength emission filters for Cy2, Cy3 and Cy5, respectively. Image alignment, spot detection, background removal and expression analysis were performed using Progenesis SameSpots (Nonlinear Dynamics, Newcastle Upon Tyne, U.K.). Fold changes and all statistical analyses were calculated based on normalized spot volumes where the internal standard was used to perform normalization. Protein Identification by Mass Spectrometry. Prior to mass spectrometry, gels were fixed for 1 h in 25% methanol, 3% orthophosphoric acid and stained overnight with Coomassie G250 (0.1% (w/v) Coomassie blue G250, 17% methanol, 2% orthophosphoric acid, 17% ammonium sulfate). Protein spots of interest were excised and destained 3 times by incubation for 5 min in 50% acetonitrile, 50 mM ammonium bicarbonate. Gel plugs were then dehydrated in 100% acetonitrile and allowed to dry completely. Proteins were digested at 37 °C overnight in 20 µL of 0.003 µg/µL porcine recombinant trypsin (Promega, Madison, WI) in 50 mM ammonium bicarbonate. Trifluoroacetic acid (TFA) was added (final concentration 0.1%), and the peptides were extracted by sonication (20 min) and subsequent desalting using C18 ZipTips (Millipore, Bedford, MA). Peptides were eluted directly into R-cyano-4-hydroxycinnamic acid matrix (5 mg/mL in 70% (v/v) acetonitrile, 0.1% TFA) onto a MALDI target plate. Peptide mass fingerprints of tryptic peptides were collected by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS) using an ABI 4700 Proteomics Analyzer, MALDI-TOF/TOF MS (Applied Biosystems, Foster City, CA,) in positive ion reflectron mode. Spectra were acquired in automated mode in the mass range of 800-4000 Da. The resulting spectra were first baseline corrected and calibrated using autodigested trypsin peaks of 842.51 and 2212.11 Da, using the DataExplorer Software Version 4.6 (UCSF, San Francisco, CA). Monoisotopic masses, [M + H]+ from the mass spectra were then extracted and analyzed using the MASCOT search engine. For peptide mass fingerprinting, protein identifications were searched and assigned using Swiss-Prot database (version 56.2, Homo sapiens containing 20 407 sequences, Swiss Institute of Bioinformatics, Basel, Switzerland) using online MASCOT version 2.2.04 [http://www.matrixscience.com]. Carbonylamidecysteine for fixed modification and oxidation of methionine residues (variable modification) were taken into account with a mass tolerance of (50 ppm. Trypsin autolysis products were excluded. One missed cleavage was allowed, except for spots 8, 28, 30, 53, 66, 80, 94 and 111 (up to 2 missed cleavages). For proteins identified based on tandem MS (MS/MS), the five most intense peptides detected in each MS mode were automatically selected for MS/MS analysis. Peptide mass (MS) and fragmentation (MS/MS) combined data were searched in Swiss-Prot database (version 50.6, H. sapiens, 14 586 sequences) using inhouse MASCOT search engine (version 1.1, Matrix Science). The search parameters were the same as for peptide mass fingerprinting mentioned above, except allowance of up to 2 missed cleavage peptides and with a MS/MS tolerance of (0.1 Da. Journal of Proteome Research • Vol. 8, No. 4, 2009 2033
research articles Proteins with MOWSE protein scores >56 (based on MS data) and >54 (based on MS and MS/MS combined data) were considered statistically significant. The following criteria were used to evaluate the search; MOWSE score, number and intensity of peptides matched, coverage of the candidate protein sequence and pI as well as molecular weight position on the 2D gel. Immunohistochemistry for Proteomic Validation and to Demonstrate Cellular Localization. Immunohistochemistry was performed on endometrial biopsies to validate the proteomic findings. Three proteins, identified as being differentially regulated between the midsecretory and midproliferative phase tissue, membrane-associated progesterone receptor component 1 (PGRMC1), Rho GDP-dissociation inhibitor 1 (Rho-GDIR) and chloride intracellular channel protein 1 (CLIC1) were chosen for localization and immunohistochemical validation. Unless stated otherwise, age range and median age were 29-43 and 37.1 for the midproliferative tissue samples and 27-38 and 31.9 for the midsecretory tissue samples. PGRMC1 was localized in the midproliferative (n ) 10) and midsecretory (n ) 10) phase tissue using an affinity purified rabbit polyclonal anti- PGRMC1 antibody (HPA002877; Atlas Antibody AB, AlbaNova University Center, Stockholm, Sweden). In brief, paraffin sections (5 µm) were dewaxed in Histosol (Sigma Chemical Co; St Louis, MO), rehydrated, and then microwaved at high power (700 W) in 0.01 mol/L sodium citrate buffer (pH 6.0) for 5 min. Endogenous peroxidase activity was quenched with 6% H2O2 in 100% methanol (1:1 v/v) for 5 min at room temperature. Nonspecific binding was blocked by incubation with nonimmune blocking solution (10% FCS/TBS) in combination with the primary antibody. Primary antibody was applied at 0.11 µg/mL for 37 °C for 60 min, and positive localization was visualized using the EnVision system and application of the peroxidise substrate 3, 3′-diaminobenzidine (DAB) (Dako, Kingsgrove, NSW, Australia), which produced a brown precipitate. Tissue sections were counterstained with Harris’ hematoxylin (Sigma Chemicals, St Louis, MO), dehydrated and mounted with DPX (ProSciTech, Thuringowa, Queensland, Australia). Negative controls were included for each section where nonimmunized rabbit IgG (Dako) was substituted at matching concentration to the primary antibody. Rho-GDIR immunohistochemistry used a mouse monoclonal antibody raised against the C-terminus of human Rho-GDIR (SC-13120; Santa Cruz Biotechnologies) with a protocol as above except that the primary antibody (0.1 µg/mL) was incubated for 30 min at 37 °C and antigen retrieval was not necessary. Negative controls used mouse IgG (Dako). CLIC1 protein was localized using a 1:100 dilution of AP823 rabbit antiserum, a gift from John Edwards.10 The immunostaining protocol was identical to that used for PGRMC1. The intensity of staining was scored for each tissue and each antibody on a scale of 0 (no staining) to 4 (maximal intense staining) in each of the cellular compartments, luminal epithelium (if available), glandular epithelium, stroma and vasculature, by two independent observers. Biological Profiling and Pathway Analysis. Differentially expressed proteins identified by MALDI-TOF/TOF MS were first investigated for their roles in biological processing. The ontology was built and assigned individually by the PANTHER (Protein Analysis through Evolutionary Relationships) Classification System [www.pantherdb.org]. The proteins were further explored and utilized as core molecules to generate biological networks through the use of Ingenuity Pathways 2034
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Chen et al. Analysis (Ingenuity Systems, Redwood city, CA, www. ingenuity.com). The scoring system of the biological and functional networks constructed is based on a hypergeometric distribution and is calculated using the right-tailed Fisher’s Exact Test. The score is the negative log of this p-value, and indicates the likelihood of the focus genes in a network being found at random. For example, a score of 6 indicates that there is a 1 in a million chance that the focus genes are together due to random chance. Therefore, networks with scores of 2 or higher are considered significant. The microarray data, which were obtained from the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) database [http://www.ncbi.nlm.nih.gov/sites/ entrez?db)geo]11 were generated from endometrial tissue on the Affymetrix GeneChip Human Genome U133 by Talbi et al.6
Results Proteomic Profile of the Mid-Secretory and Mid-Proliferative Endometrium. 2D DIGE was used to comparatively assess protein expression in eutopic endometrial tissue in either the midproliferative (days 8-12) or midsecretory (days 19-23) phase of the menstrual cycle. A total of 1017 protein spots were resolved across a pH 4-7 range (Figure 1). Experimental variability was assessed using the Cy2 internal standard present in every gel. An average coefficient of variation (CV) of 21% was observed for the replicate samples across the data set, demonstrating good technical reproducibility. Biological replicates of samples derived from the proliferative (n ) 4) or secretory phase (n ) 4) showed a CV in normalized volume of 23% and 36%, respectively. Only those proteins that showed significant (p < 0.05) changes in protein expression across all individuals in the group were considered for further analysis. When compared to eutopic tissue in the proliferative phase of the cycle, a total of 39 protein spots showed a significant (p < 0.05) decrease in expression (-3.7- to -1.5-fold), while a further 157 showed an increase in the midsecretory phase. To ensure that the observed changes were not due to artifacts of the labeling procedure, a reciprocal labeling experiment was undertaken using pooled secretory phase samples. No dyespecific change in any of the identified proteins was observed (data not shown). Differentially expressed proteins were then excised from the gel and identified by MALDI-TOF/TOF MS (Tables 1 and 2). Protein isoforms that were identified in multiple spots are grouped together in Table 1, and are ranked according to their pI’s (from acidic to basic) as identified in the 2D gel (Figure 1). Of the 196 spots interrogated (p < 0.05), 76 were identified yielding 41 unique gene products. Of the 76 proteins identified, 8 spots were also found to contain mixtures of more than one protein (Table 2). Since it was not possible to assign the fold change unambiguously to these protein mixtures, only changes in normalized spot volume of the mixture are indicated (Table 2). A total of 14 proteins were identified as multiple isoforms that differed in isoelectric point and/or molecular weights (Table 1, Figure 1). For example, spots 42, 39, 29 and 38 were found to be isoforms of the protein, serum albumin precursor (ALBU), which had the same molecular weight but exhibited different isoelectric points (pI’s) ranging from around 5.5 to 5.9. Heat shock protein beta-1 (HSPB1) had four isoforms (spot 50, 128, 157 and 88), which exhibited differences in their apparent molecular weights as well as their pIs (Figure 1). The failure to identify the remaining 120 proteins by mass spectrometry is due to the limited sensitivity of the mass spectro-
Cyclical Changes in Endometrial Proteome
research articles
Figure 1. 2D SDS PAGE image of endometrial tissue proteins. Proteins (150 µg) extracted from human endometrial tissue were separated by 2D PAGE (first dimension pH 4-7, second dimension 8-16% polyacrylamide gradient). A representative gel of the pooled internal standard (Cy2) is shown. Differentially expressed proteins identified by MALDI-TOF/TOF MS are numbered and indicated by circles.
metric method compared to the fluorescent dye labeling, image detection and analysis procedures. When comparing between the midproliferative and midsecretory phases, marked differences in expression changes were observed between isoforms of the same proteins. For example, three isoforms of Annexin A4 (ANXA4) were found to increase from 2.4-fold (spot 55; Figure 1) to 3.7-fold (spot 9; Figure 1) from the midproliferative to midsecretory phase (Table 1). Similarly, 7 different isoforms of Keratin 8 (K2C8) and 4 isoforms of HSPB1 were found to increase from 2.4- to 3.8-fold and 1.6- to 2.4-fold, respectively, in the midsecretory phase. By contrast, 4 isoforms of albumin ALBU (4 isoforms, fold change range -2.6 to -2.9) showed decreased expression across the cycle ranging between 2.6- to 2.9-fold. These results suggest the presence of post-translational modifications of proteins during the transition from proliferative to secretory phase (Table 1). Validation of Selected Proteins. To validate the changes in protein levels observed in this proteomic analysis, and to determine the cellular location of the proteins, immunohistochemistry was performed on 20 individual tissue sections, representing the midproliferative (n ) 10) and midsecretory (n ) 10) phases of the cycle. Three representative proteins, PGRMC1, Rho-GDIR and CLIC1, were chosen based on their biological functions and antibody availability. Immunostaining revealed that PGRMC1 was confined only in the stromal compartment with stronger staining in the proliferative phase
tissue (Figure 2A,B), although the intensity of staining was variable across tissue samples, ranging from undetectable to moderately strong as demonstrated in Figure 3A. No immunoreactive PGRMC1 was detected in the epithelial compartment or in the vasculature. The decreased expression of PGRMC1 in the secretory phase agreed with the 3.1- to 3.7fold decrease in expression observed for two identified isoforms (spots 10 and 24) of PGRMC1 from proteomic data (Figure 3A). This suggests that processing of the two isoforms of PGRMC1 is consistent between the midproliferative and midsecretory cycle. Immunostaining of Rho-GDIR showed strong staining of glands where it was primarily localized to the apical surface. Immunoreactive staining of glandular Rho-GDIR was significantly elevated (4.8-fold p ) 0.0002) in secretory phase tissue, and was not detectable in the majority (8 of 10, Figure 3C) of the midproliferative phase tissues. By contrast, staining in the luminal epithelium was not detectable in all but one tissue sample in the midsecretory phase. A low level staining was also observed in the stromal compartment which did not differ significantly between phases of the cycle. The increased expression of Rho-GDIR observed in the apical surface of glands is in agreement with proteomic data, where two isoforms of Rho-GDIR identified (spots 91 and 62) each showed increased expression in the midsecretory phase (Figure 3D). Immunostaining revealed that CLIC1 was only detected in epithelial cells, where both glandular and luminal staining Journal of Proteome Research • Vol. 8, No. 4, 2009 2035
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Table 1. Differentially Expressed Proteins Identified by MALDI-TOF /TOF MS published mRNA microarray analysis6
proteomic analysis spot no.
Swiss-Prot accession ID
86 30 166
1433E_HUMAN 1433S_HUMAN 2AAA_HUMAN
167
ABHEB_HUMAN
114
ACTB_HUMAN
147 42 39 29 38 120 55 9 18 276 170 485 99
ACTG_HUMAN ALBU_HUMAN ALBU_HUMAN ALBU_HUMAN ALBU_HUMAN ANXA1_HUMAN ANXA4_HUMAN ANXA4_HUMAN ANXA4_HUMAN ANXA5_HUMAN ANXA5_HUMAN CALD1_HUMAN CLIC1_HUMAN
protein description
fold changea
MOWSEb
14-3-3 protein epsilon 14-3-3 protein sigma Serine/threonine-protein phosphatase 2A 65 kDa regulatory subunit A alpha isoform Abhydrolase domain-containing protein 14B Actin, cytoplasmic 1
2.1 2.9 -1.5
72 74 59 MS/MSe
128
Actin, cytoplasmic 2 Serum albumin precursor Serum albumin precursor Serum albumin precursor Serum albumin precursor Annexin A1 Annexin A4 Annexin A4 Annexin A4 Annexin A5 Annexin A5 Caldesmon Chloride intracellular Chloride intracellular channelchannel protein 1 CLIC1_HUMAN Chloride intracellular Chloride intracellular channelchannel protein 1 CNDP2_HUMAN Cytosolic nonspecific dipeptidase CNDP2_HUMAN Cytosolic nonspecific dipeptidase CNDP2_HUMAN Cytosolic nonspecific dipeptidase CNDP2_HUMAN Cytosolic nonspecific dipeptidase COTL1_HUMAN Coactosin-like protein DC1I2_HUMAN Cytoplasmic dynein 1 intermediate chain 2 ENOA_HUMAN Alpha-enolase EZRI_HUMAN Ezrin PDIA3_HUMAN Protein disulfide-isomerase A3 precursor GANAB_HUMAN Neutral alpha-glucosidase AB precursor GANAB_HUMAN Neutral alpha-glucosidase AB precursor GDIR1_HUMAN Rho GDP-dissociation inhibitor 1 GDIR1_HUMAN Rho GDP-dissociation inhibitor 1 GDIR2_HUMAN Rho GDP-dissociation inhibitor 2 GRP75_HUMAN Stress-70 protein, mitochondrial precursor GSHB_HUMAN Glutathione synthetase RB11B_HUMAN Ras-related protein Rab-11B HNRH3_HUMAN Heterogeneous nuclear ribonucleoprotein H3 HSP71_HUMAN Heat shock 70 kDa protein 1 HSP71_HUMAN Heat shock 70 kDa protein 1 HSP71_HUMAN Heat shock 70 kDa protein 1 HSPB1_HUMAN Heat shock protein beta-1 (Heat shock 27 kDa protein) HSPB1_HUMAN Heat shock protein beta-1
157 88 142
HSPB1_HUMAN HSPB1_HUMAN IDHC_HUMAN
74
72 22 53 15 7 66 126 69 105 25 48 37 91 62 1 8 186 75 111 84 43 162 50
1.5
61
1.9
182 MS/MSe 66 51 82 76 71 105 107 130 115 98 58 60 76
1.6 -2.6 -2.7 -2.9 -2.7 1.8 2.4 3.7 3.3 1.7 1.5 1.3 2.0 2.2 3.2 2.4 3.4 3.7 2.3 -1.8
microarray fold changea
14 10 10
54 31 27
1.1 3.5 -1.2
6
37
1.1
11
43
-1.0
8 14 23 21 25 22 16 24 24 14 10 13 12
28 22 34 28 30 57 42 58 63 43 31 21 43
-1.0 -1.1 -1.1 -1.1 -1.1 2.0 4.4 4.4 4.4 -1.2 -1.2 1.1 1.6
8
29
1.6
17 11 25 18 7 4
39 24 61 44 38 10
1.8 1.8 1.8 1.8 -1.0 1.3
19 30 23
39 47 50
1.1 3.1 1.0
-2.5
89
12
14
-1.3
-2.7
60
22
22
-1.3
2.1 2.4 6.5 3.7
62 68 63 58
8 11 9 17
37 45 49 40
1.9 1.9 -1.0 -1.1
1.4 2.2 1.9
99 57 60
20 7 7
51 36 31
-1.5 -1.2 -1.1
2.1 2.6 1.6 2.4
99 95 84 59
19 14 13 8
33 32 22 48
3.7 3.7 3.7 1.4
1.8
79 MS/MSe 73 84 80
4
28
1.4
6 14 23
36 64 46
1.4 1.4 1.3
13
28
1.0
27 25 18 7 34 35 45
60 53 40 14 63 64 68
1.7 1.7 1.7 -1.1 -1.1 1.4 1.4
2.3 2.0 3.1
1.6 2.1 1.7
IFT81_HUMAN
85 23 47 192 27 34 17
K1C18_HUMAN K1C18_HUMAN K1C18_HUMAN K2C1_HUMAN K2C7_HUMAN K2C8_HUMAN K2C8_HUMAN
Keratin, Keratin, Keratin, Keratin, Keratin, Keratin, Keratin,
2.1 3.2 2.5 1.4 3.1 2.8 3.4
2036
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I cytoskeletal 18 I cytoskeletal 18 I cytoskeletal 18 II cytoskeletal 1 II cytoskeletal 7 II cytoskeletal 8 II cytoskeletal 8
% coveraged
113 67 88 81 66 55 MS/MSe 73 92 88
Heat shock protein beta-1 Heat shock protein beta-1 NADP-dependent isocitrate dehydrogenase Intraflagellar transport 81 type type type type type type type
79
no. of matched peptidesc
2.2
55 MS/MSe 216 190 123 61 196 211 192
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Cyclical Changes in Endometrial Proteome Table 1. Continued
published mRNA microarray analysis6
proteomic analysis spot no.
Swiss-Prot accession ID
6 13 12 61 21 94 97 100 184 24
K2C8_HUMAN K2C8_HUMAN K2C8_HUMAN K2C8_HUMAN K2C8_HUMAN KLH20_HUMAN MVP_HUMAN MVP_HUMAN NPM_HUMAN PGRC1_HUMAN
10
PGRC1_HUMAN
127 28 45 80
SORCN_HUMAN TPM2_HUMAN TPM4_HUMAN TPM4_HUMAN
no. of matched peptidesc
% coveraged
microarray fold changea
204 118 136 157 125 59 225 102 63 109
44 24 36 31 25 15 36 19 7 7
67 46 65 52 53 27 38 29 34 28
1.4 1.4 1.4 1.4 1.4 -1.2 3.6 3.6 -1.4 -2.7
-3.7
69
7
28
-2.7
1.8 2.9 2.5 2.2
69 74 59 96
7 14 10 161
31 48 29 58
2.1 -1.1 -1.4 -1.4
protein description
fold changea
Keratin, type II cytoskeletal 8 Keratin, type II cytoskeletal 8 Keratin, type II cytoskeletal 8 Keratin, type II cytoskeletal 8 Keratin, type II cytoskeletal 8 Kelch-like protein 20 Major vault protein Major vault protein Nucleophosmin Membrane-associated progesterone receptor component 1 Membrane-associated progesterone receptor component 1
3.8 3.5 3.6 2.4 3.2 -2.0 2.0 2.0 1.4 -3.1
Sorcin Tropomyosin beta chain Tropomyosin alpha-4 chain Tropomyosin alpha-4 chain
MOWSEb
a A positive fold change indicated that protein/gene expression was up-regulated in the secretory phase. A negative fold change indicated that protein/ gene expression was up-regulated in the proliferative phase. b MOWSE protein score based on MS data. In all cases, a probability score
