Proteomics in the Diagnosis of Endometriosis: Opportunities and Challenges

The non‐surgical diagnosis of endometriosis is still challenging for the clinician. Ultrasonography and magnetic resonance imaging can be used to diagnose ovarian endometriotic cysts and deep infiltrating endometriosis; but their performance is poor in the diagnosis of initial stages of endometriosis. CA‐125 and other serum markers (such as CA 19‐9, serum protein PP14, interleukins, and angiogenetic factors) have been measured in women with endometriosis but they are not reliable for the diagnosis of the disease. Although several studies used proteomics technologies to identify plasmatic markers of endometriosis, the non‐invasive diagnosis of endometriosis is far from being achieved. In this issue, Manousopoulou et al. compare the integrated quantitative proteomic profile of eutopic endometrium and serum of women with endometriosis and controls. 1214 proteins are differentially expressed in the eutopic endometrium and 404 proteins in the serum of the two study groups. 21 proteins are aberrantly expressed in both eutopic endometrium and serum of women with endometriosis. More work is needed to assess if the differentially expressed proteins identified in this study can be used as clinical markers of endometriosis.     

Understanding the pathogenesis of endometriosis through proteomics: Recent advances and future prospects

Endometriosis is a complex gynecological disease, characterized by the presence and growth of endometrial tissue outside the uterus, resulting in pelvic pain and infertility. It occurs in 10% of women in their reproductive age. The viable endometrial cells enter the peritoneal cavity by retrograde menstruation, implant, and cause lesions ectopically; depending on their ability to survive, attach, grow, and invade. These “normal” endometrial cells turn “endometriotic” apparently because of inherent abnormalities present in them. Information on these molecular abnormalities is now being sought through proteomic approaches. Recent proteome-based comparisons between the eutopic endometrium from normal women and patients with endometriosis have revealed several proteins (many of which are shown to have a role in several cancers), of which a few have been validated as potential players in the etiology of endometriosis. After an initial in-flow of information from these proteome studies of eutopic endometrium, focus now needs to be expanded to the changes in the various protein PTMs and their upstream effectors present in these tissues. Early diagnosis of endometriosis through noninvasive means is the need of the hour as well—which would require the use of the presently existing immunoassays, along with the advancing MS-based proteomics. In this review, we aim to discuss these future thrust areas of human endometriosis proteomics and also present the proteomic advances made so far in understanding the molecular basis of endometriosis.     

Altered proteins of the anterior cingulate cortex white matter proteome in schizophrenia

Alterations in the circuitry between and within different brain regions including the anterior cingulate cortex (ACC) is implicated in the neuropathology of schizophrenia. The involvement of white matter in schizophrenia is becoming increasingly apparent with reports of structural, morphological and genetic alterations occurring in the disease. The 2‐DE was employed to reveal significantly altered proteins within the ACC white matter proteome in a schizophrenia cohort (n = 10) relative to controls (n = 10). From the 423 matched spots between the two groups, the levels of 32 protein spots were altered in the schizophrenia ACC white matter. Of these, 30 spots were identified using MS. The majority of the altered proteins in schizophrenia function in metabolism, the cytoskeleton, and the synapse. These proteomic data suggest that the brain circuitry involving the ACC white matter tracts is altered in schizophrenia, possibly caused by depleted glucose metabolism and altered structural components of this region.     

Anatomic site‐specific proteomic signatures of gastrointestinal stromal tumors

The gastrointestinal stromal tumor (GIST) is the most common mesenchymal malignancy of the gastrointestinal tract. Its clinical course ranges widely from a curable disorder to a highly malignant disease. Although its clinical and molecular characteristics depend on the anatomic site of origin, the molecular background of GIST arising in different anatomical site has not been studied yet. To investigate the proteomic background of GIST, we examined the proteomic features corresponding to the anatomic site of tumor origin. Comparison of the proteomic profile of gastric (23 cases) and small intestinal (9 cases) GIST by 2‐DE revealed 105 protein spots with significantly different intensity (p <0.01) between the two groups. Mass spectrometric study identified 68 distinct proteins for these 105 protein spots, including cancer‐associated ones such as prohibitin, pigment epithelium‐derived factor, and alpha‐actinin 4. The intensity of 37/105 (35.2%) protein spots was significantly concordant with the corresponding mRNA levels (p <0.01). Although both 2‐D DIGE and microarray experiments showed significant up‐regulation of vimentin expression in small intestinal GIST, Western blotting did not show a significant difference between the two groups. In conclusion, our study demonstrates the proteins specially expressed in GIST depending on their site of origin, as well as the unique advantage offered by use of proteomics to acquire such data. The identified proteins may provide clues to understanding the different characteristics of GIST depending on their site of origin.     

Proteome of human endometrium: Identification of differentially expressed proteins in proliferative and secretory phase endometrium

Purpose: To exploit the potential of proteomics to identify and study additional yet‐unidentified important proteins present in human endometrium. Experimental design: The proteome of human endometrium would be established using 2‐DE and MALDI and the data analyzed to identify differential protein expression in the proliferative and secretory phase of the menstrual cycle using PDQuest software and MALDI. Results: In the present work, 2‐DE of human endometrium protein led to the resolution of over 200 spots. Subsequent MALDI analysis of 215 spots allowed the identification of 194 proteins. A total of 57 out of the 215 spots were found to be differentially expressed, out of which 49 could be identified using MALDI. These differentially expressed proteins included structural proteins, molecular chaperones, signaling proteins, metabolic proteins, proteins related to immunity, RNA biogenesis, protein biosynthesis and others. The differential expressions of seven representative proteins in secretory and proliferative phase endometrium tissue were confirmed by immunoblot analysis. Conclusion and clinical relevance: This study establishes the 2‐D proteome of human endometrium represented by 194 identified protein spots. The present data provides an important clue towards determining the function of these proteins with respect to endometrium related diseases.     

Characterization of molecular markers indicative of cervical cancer progression

Cervical cancer originates with human papillomavirus (HPV) infection and progresses via histologically defined premalignant stages. Here we compare normal cervical epithelium and patient‐matched high‐grade squamous intraepithelial lesions (HSIL) with cervical carcinoma tissue from the same patient population (n = 10 per group). Specimens were analyzed by combined laser capture microdissection and 2‐D DIGE. Significant expression changes were seen with 53 spots resulting in identification of 23 unique proteins at the molecular level. These include eight that uniquely distinguish normal epithelium and HSIL and four that uniquely distinguish HSIL and carcinoma. In addition, one protein, cornulin, distinguishes all three states. Other identified proteins included differentiation markers, oncogene DJ‐1, serpins, stress and interferon‐responsive proteins, detoxifying enzymes, and serum transporters. A literature review, performed for all identified proteins, allowed most changes to be assigned to one of three causes: direct or indirect HPV oncoprotein interactions, growth selection during latency, or interactions in the lesion microenvironment. Selected findings were confirmed by immunohistochemistry using either frozen sections from the same cohort or formalin fixed paraffin embedded samples from a tissue microarray. Novel markers described here have potential applications for increasing the predictive value of current screening methods.     

Diagnostic protein marker patterns in squamous cervical cancer

Purpose: Cervical cancer is the second most prevalent malignancy of women. Our aim was to identify additional marker protein patterns for objective diagnosis of squamous cervical cancer (SCC). Experimental design: Collected tissue biopsies of SCC, squamous vaginal cancer (SVC), normal cervical and vaginal mucosa were subjected to 2‐DE, SameSpot analysis, MALDI‐TOF‐MS protein identification, and analysis of the expression of selected proteins by immunohistochemistry. Results: In 148 protein spots selected by the difference in expression 99 proteins were identified. A differential protein pattern for SCC was, e.g. over‐expressed (OE) eukaryotic translation initiation factor 3‐2β, neutrophil cytosolic factor 2, annexin A6 (ANXA6), for SVC it was OE cathepsin D, γ‐catenin, RAB2A, for both cancers it was OE apolipoprotein E, tropomyosin 3, HSPA8, and underexpressed cytokeratin 13, osteoglycin. In SCC nuclear expression of neutrophil cytosolic factor 2, PRDX2, HSP27 (nine of ten cases), ANXA6 (nine of ten cases) was observed while tropomyosin 4 was expressed only in two of ten cases. There was 81.1% (43/53) agreement between the expression of protein spots and the immune expression of proteins ( www.proteinatlas.org ). Conclusions and clinical relevance: SCC is characterized by specific tissue marker protein patterns that allow objective detection of the disease. They can become a basis for objective automated cytology‐based screening and improve current diagnostics of SCC.     

In pursuit of B-cell synovial autoantigens in rheumatoid arthritis: Confirmation of citrullinated fibrinogen, detection of vimentin, and introducing carbonic anhydrase as a possible new synovial autoantigen

We aimed to investigate potential synovial autoantigens in rheumatoid arthritis (RA) that could trigger the induction of B‐cell autoantibodies. Total protein extract of synovial tissue obtained from seven RA patients was pooled and separated by 1‐DE and 2‐DE. The corresponding blots were probed with sera from RA (n = 30) and disease control samples (n = 30). Protein spots showing a sensitivity of >15% were identified by MS. 1‐D immunoblots revealed one protein band with a specificity in RA of 100%, a sensitivity of 43%, which was identified as fibrinogen β chain. 2‐D analysis revealed the subunits of fibrinogen, especially the β and γ chain, as the most prominent synovial autoantigens. We also identified vimentin, the Sa‐antigen and carbonic anhydrase I as a potentially new synovial autoantigen. The protein patterns of these immunoreactive spots were observed as trains. The spots showing the highest autoimmune reactivity occurred at the acidic side of these trains and were recognized by anticitrullinated protein/peptide antibodies positive RA sera. Antimodified citrulline staining of these patterns confirmed protein citrullination. Therefore, PTMs such as citrullination due to alterations of peptidylarginine deiminase activity or generation of RA‐specific epitopes, should be considered as a trigger in tolerance break.     

Prostate cancer serum biomarker discovery through proteomic analysis of alpha‐2 macroglobulin protein complexes

Alpha‐2 macroglobulin (A2M) functions as a universal protease inhibitor in serum and is capable of binding various cytokines and growth factors. In this study, we investigated if immunoaffinity enrichment and proteomic analysis of A2M protein complexes from human serum could improve detection of biologically relevant and novel candidate protein biomarkers in prostate cancer. Serum samples from six patients with androgen‐independent, metastatic prostate cancer and six control patients without malignancy were analyzed by immunoaffinity enrichment of A2M protein complexes and MS identification of associated proteins. Known A2M substrates were reproducibly identified from patient serum in both cohorts, as well as proteins previously undetected in human serum. One example is heat shock protein 90 alpha (HSP90α), which was identified only in the serum of cancer patients in this study. Using an ELISA, the presence of HSP90α in human serum was validated on expanded test cohorts and found to exist in higher median serum concentrations in prostate cancer (n = 18) relative to control (n = 13) patients (median concentrations 50.7 versus 27.6 ng/mL, respectively, p = 0.001). Our results demonstrate the technical feasibility of this approach and support the analysis of A2M protein complexes for proteomic‐based serum biomarker discovery.     

Protein-induced changes during the maturation process of human dendritic cells: A 2-D DIGE approach

Dendritic cells (DCs) are unique antigen presenting cells, which upon maturation change from a specialized antigen‐capturing cell towards a professional antigen presenting cells. In this study, a 2‐D DIGE analysis of immature and mature DCs was performed, to identify proteins changing in expression upon maturation. The protein expression profile of immature and mature DCs, derived from CD14⁺ peripheral blood monocytes was investigated using two pH ranges (pH 4–7 and 6–9) (n = 4). Ninety one differentially expressed spots (p<0.01) were detected, from which we identified 74 spots (81.32%) corresponding to 41 different proteins. The proteins identified play a role in diverse processes, such as antigen processing/presentation, vesicle transport and cytoskeleton remodeling. In addition, a protein interaction network contained 29 (out of 41) proteins, suggesting that, although they functionally originate from distinct classes, these proteins are acting as a protein‐interactome. In conclusion, the proteins shown here to be altered in expression upon maturation are in line with the morphological and functional changes observed during the maturation process, providing a better understanding of the processes involved. This will open new avenues for investigating treatment regimens for immune‐associated disorders.     

Characterisation of tumoral markers correlated with ErbB2 (HER2/Neu) overexpression and metastasis in breast cancer

The receptor tyrosine kinase ErbB2 (HER2/neu) is overexpressed in ?30% of breast cancers and is associated with poor prognosis and an increased likelihood of metastasis. Clinical treatments such as trastuzumab are effective in less than 35% of women diagnosed as ErbB2‐positive, highlighting the necessity of searching for novel targets and alternative therapies. Herein, a proteomic screening strategy combining quantitative‐based gel electrophoresis and MS was used to compare the protein expression of 48 normal human breast and tumour tissues differing in ErbB2 expression and lymph node status. The aim was to identify proteins associated with the aggressive phenotype of ErbB2‐positive breast cancer which could be potential biomarkers of the disease as well as therapy targets. In total, 177 protein isoforms (107 gene products) differentially expressed between tissue groups were identified. Immunohistochemical staining of a tissue‐microarray was used for validation of selected protein candidates. We found that expression of HSP90α, laminin and GSTP1 significantly correlated with ErbB2 expression, while others such as AGR2, NM23H1 and Annexin 2 were overexpressed in greater than 40% of tumours. Finally, knocking‐down the expression by RNA interference of three candidates, AGR2, Transgelin2 and NM23H1 resulted in an enhanced invasive capacity of MDA‐MB435 cells. These data support the involvement of these targets in tumour progression and identify them as novel biomarkers of the disease.     

Spontaneous and induced labour are associated with different myometrial proteomes in the human

Human myometrium undergoes a major phenotypic change at labour likely involving modifications to key regulatory proteins. In some cases, the myometrium fails to activate normally and medical intervention is required to induce labour. In this study, 2‐D DIGE was used to examine changes in the myometrial proteome at the time of spontaneous (SL) and induced labour (IL). Proteomic profiles of nonlabouring term myometria (NL, n = 6) were quantitatively compared to SL (n = 6) and prostaglandin/oxytocin‐IL term myometria (n = 6). In SL samples, 23 differentially expressed protein spots were detected (9 increased/14 decreased compared to NL, p<0.05). In IL samples, 59 differentially expressed spots were observed (13 increased/46 decreased compared to NL). Comparison of SL and IL proteomes revealed 69 differentially expressed proteins (7 increased/62 decreased). Two proteins consistently decreased in SL and IL samples were identified as transgelin (1.98‐ and 1.97‐fold decrease in SL and IL, respectively) and αB‐crystallin (3.27‐ and 2.49‐fold decrease). Levels of desmin and cytosolic phospholipase A2 β were decreased 2.9‐ and 2.65‐fold, respectively only in IL samples. Our results show human labour is accompanied by general downregulation of specific myometrial proteins. Differences exist between SL and IL myometrial proteomes indicating divergence of underlying processes and highlighting the importance of distinguishing these groups in future studies of parturition. Our findings underscore the utility of discovery approaches in investigations of organ‐wide protein changes that underlie discrete physiological events including human labour.     

Plasma proteome of buffaloes

The proteomic approach has aroused the interest of veterinary medicine researchers, especially regarding the production of biopharmaceuticals and diagnosis of diseases in farm animals. Water buffaloes have gained prominence in the world economy due to the quality of their milk, meat, and leather, in addition to being an important donor of blood components. This work aimed to identify and characterize the proteins present in the blood plasma of Murrah buffaloes (Bubalus bubalis) through 2D electrophoresis, in gel protein digestion followed by mass spectrometry technique and for albumin depletion, in solution protein digestion followed by shotgun analysis. Our results showed the identification of 112 protein spots and 35 individual proteins, respectively. The abundant proteins were represented by albumin, fibrinogen‐α, fibrinogen‐β, fibrinogen‐γ, immunoglobulins in general, α‐1‐antiproteinase, α‐1B‐glycoprotein, α‐2‐HS‐glycoprotein, α‐macroglobulin, apolipoprotein A1, antithrombin‐III, endopin 2B, fetuin‐B, retinol‐binding protein, serotransferrin, transthyretin and vitamin D‐binding protein. Most of these proteins are related to the signaling pathways of the complement system and coagulation cascade. The results allowed a better understanding of the protein composition of these blood components, thus promoting studies on animal health in the search for molecular markers of zoonotic diseases in buffaloes.     

Proteomic analysis of the response of the human neutrophil-like cell line NB-4 after exposure to anthrax lethal toxin

We used 2‐D DIGE to analyze the early response of NB‐4 cells, a human promyelotic leukemia cell line, exposed to lethal toxin from Bacillus anthracis at the proteome level. After a 2 h exposure, cells were still viable and 43% of spots (n = 1042) showed a significant change in protein level. We identified 59 spots whose expression had changed significantly, and these reflected cytoskeleton damage, mitochondrial lysis and endoplasmic reticulum stress. Actin filament assembly was disrupted as evidenced by an increase in both actin subunits and phosphorylated cofilin, whilst levels of tropomyosin, tropomodulin and actin related protein 2/3 complex subunit decreased. Lower levels of ATP synthase subunits and mitochondrial inner membrane protein were identified as markers of mitochondrial lysis. Levels of various stress response proteins rose and, uniquely, levels of Ca²⁺ binding proteins such as translationally controlled tumor protein rose and hippocalcin‐like protein 1 decreased. This response may have mitigated effects brought about by mitochondrial lysis and endoplasmic reticulum stress, and delayed or prevented apoptosis in NB‐4 cells. These results resemble findings of similar proteomics studies in murine macrophages, although quantitative differences were observed.     

Toward the Proteome of the Human Peripheral Blood Eosinophil

Eosinophils (EOSs) are granular leukocytes that have significant roles in many inflammatory and immunoregulatory responses, especially asthma and allergic diseases. We have undertaken a fairly comprehensive proteomic analysis of purified peripheral blood EOSs from normal human donors primarily employing 2‐DE with protein spot identification by MALDI‐MS. Protein subfractionation methods employed included IEF (Zoom^® Fractionator) and subcellular fractionation using differential protein solubilization. We have identified 3141 proteins, which had Mascot expectation scores of 10^?3 or less. Of these 426 were unique and non‐redundant of which 231 were novel proteins not previously reported to occur in EOSs. Ingenuity Pathway Analysis showed that some 70% of the non‐redundant proteins could be subdivided into categories that are clearly related to currently known EOS biological activities. Cytoskeletal and associated proteins predominated among the proteins identified. Extensive protein posttranslational modifications were evident, many of which have not been previously reported that reflected the dynamic character of the EOS. This data set of eosinophilic proteins will prove valuable in comparative studies of disease versus normal states and for studies of gender differences and polymorphic variation among individuals.     

Screening of serum samples from Wegener's granulomatosis patients using antibody microarrays

Wegener's Granulomatosis (WG) is an idiopathic granulomatosis autoimmune vasculitis that primarily affects small vessels and is associated with glomerulonephritis and pulmonary granulomatous vasculitis. Anti‐neutrophil cytoplasmic auto‐antibodies (cANCA) against proteinase‐3 are used to identify WG, but ANCA titers are not present in some patients with the localized disease. The objective of this study was to develop an antibody array to help identify protein expression patterns in serum from patients with WG as compared to normals. The arrays were tested for limits of detection, background, and cross reactivity using standard proteins. The arrays were hybridized with either normal patient serum (n = 30) or with serum samples from a population of WG patients (n = 26) that were age and sex matched. Data analysis and curve fitting of the standard dilution series calculated r² values and determined a sensitivity of <50 pg/mL for the majority of proteins. A total of 24 proteins were assessed. Several statistically significant increases (p<0.05) were seen in the expression of: angiotensin converting enzyme‐I, IFN‐γ, IL‐8, s‐ICAM‐1 and s‐VCAM in WG patients as compared to controls. Utilizing the antibody microarray technology has led to the identification of potential biomarkers of vascular injury in the serum of WG patients.     

The murine plasma protein response to polymicrobial intra-abdominal sepsis

Protein biomarkers in the peripheral blood could potentially be used as early indicators of sepsis and a means to stratify patients for clinical trials. Although individual molecular markers have been proposed for sepsis, none has clinical utility. The global changes in plasma proteins over the clinical course of sepsis have not been characterized using proteomic methods. We used cecal ligation and puncture to induce polymicrobial sepsis in mice and generated plasma protein profiles using 2‐D DIGE of plasma from septic mice and surgical controls. Replicate cohorts (n = 3) of 4–7 animals each were used to identify 62 gel features that changed significantly (Student's t‐test, p<0.05). We identified a suite of plasma proteins that describe uniquely the host plasma response to polymicrobial septic insult. Principal components analysis of protein abundance showed that ～90% of the variability between samples was due to sepsis. In addition to canonical acute phase proteins, we identified proteins that are associated with metabolic changes (e.g. α‐2 HS glycoprotein and zinc α‐2 glycoprotein) consistent with the pathophysiology of sepsis. The panel of sepsis‐associated molecular markers identified herein may prove useful in the diagnosis and categorization of sepsis.     

Quantification of Biomarker Proteins Using Reverse‐Phase Protein Arrays

It is expected that antibody‐based proteomics will soon occupy a pivotal position in the discovery and validation of biomarkers and therapeutic targets. The reverse‐phase protein array (RPPA) is an antibody‐based proteomic method that can quantify the expression of multiple posttranslationally modified proteins (such as those that have been phosphorylated) across a large number of protein samples. RPPA is highly sensitive and requires only very small protein samples. This feature, in combination with large antibody libraries, makes RPPA ideal for clinical proteomics, as well as the fact that it is an expandable multiplex assay. In Volume 14, Issue 1 of Proteomics Clinical Applications, Suzuki and colleagues report for the first time a study comparing RPPA and immunohistochemistry for quantification of seven biomarker proteins used for subtyping of diffuse large B‐cell lymphoma. Such combination of multiple biomarkers is likely to increase diagnostic accuracy and can be used for precise classification of this heterogeneous disease.     

Proteomic Helicobacter pylori biomarkers discriminative of low-grade gastric MALT lymphoma and duodenal ulcer

To date no reliable diagnostic method exists to predict, among the very large and clinically heterogeneous group of Helicobacter pylori‐infected patients, the extremely small group at risk for developing low‐grade gastric MALT lymphoma (LG‐MALT). Search of proteomic biomarkers holds promise for the classification of the H. pylori strains with regard to this severe clinical outcome. In the present study 69 H. pylori strains isolated from patients with two different H. pylori‐associated diseases, duodenal ulcer (DU, n=29) and LG‐MALT (n=40) were used. Protein expression patterns of the strains were analyzed by using the high‐throughput methodology SELDI. Selected proteins were purified by means of chromatographic and electrophoretic methods in view of further sequencing by LC‐MS/MS. Univariate analysis (Mann–Whitney test) of the protein expression patterns generated nine significant biomarkers that can discriminate between H. pylori strains from patients with DU and LG‐MALT. These biomarkers are of low molecular weight, ranging from 6 to 26.6 kDa. Among them, two are overexpressed in LG‐MALT strains and seven – in DU strains. Two biomarker proteins, one overexpressed in LG‐MALT strains (13.2 kDa) and another one – overexpressed in DU strains (26.6 kDa), were purified to homogeneity and identified by using LC‐MS/MS as a 50S ribosomal protein L7/L12 and a urease subunit, respectively. These biomarkers can be included in novel protein arrays for the differential diagnosis of H. pylori‐associated clinical outcomes.