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.     

Proteomic profiling of differential display analysis for human oral squamous cell carcinoma: 14-3-3 σ Protein is upregulated in human oral squamous cell carcinoma and dependent on the differentiation level

Oral squamous cell carcinoma (OSCC) has an absolute majority of all oral cancer. We used proteomic technology to analyze the protein expression profile in OSCC tissues and accompanying surrounding normal tissues in four oral locations (buccal mucosa, gingival mucosa, oral floor, and tongue). Ten protein spots were overexpressed more strongly in cancer tissues than normal ones, and were identified as proliferating cell nuclear antigen, 14-3-3 ε, 14-3-3 σ, proteasome subunit α type 5, translationally controlled tumor protein, eukaryotic translation initiation factor 3 subunit, macrophage capping protein, and mitochondrial isocitrate dehydrogenase subunit α. Macrophage capping protein and mitochondrial isocitrate dehydrogenase subunit α had two spots. Especially, we focused on 14-3-3 σ protein, one of the eight identified proteins, and assessed its expression level in four oral locations of OSCC by using differential display methods. The expression level of 14-3-3 σ protein was upregulated in four locations of oral cavity. Eight proteins which we identified in this study may play an important role in OSCC carcinogenesis and progression and could be used as diagnostic biomarkers of OSCC.     

Analysis of the differential proteome of human erythroblasts during in vitro erythropoiesis by 2-D DIGE

In the present study we have used an in vitro culture system that induces differentiation of human CD34(+) cells down the erythroid lineage along with 2-D DIGE to determine the differential proteome of erythroblasts at specific developmental stages during erythropoiesis. We initially distinguished 154 proteins differentially expressed between pro-normoblasts and polychromatic/orthochromatic erythroblasts, of which 24 protein spots, representing 21 different proteins, were identified following MS/MS and verification in replicate experiments with cells from different individuals. These data were confirmed by analysis of the differential proteome of erythroblasts at more discrete stages of erythropoiesis using 2-D DIGE and by mapping the expression of three identified proteins (Annexin I, Annexin II, Carbonic Anhydrase I) throughout erythropoiesis by Western blot with specific antisera. In addition, the differential expression of proteins due to biological variation, such as polymorphism, was determined by comparing erythroblasts at the same developmental stage from different individuals; none of the proteins thus identified were represented in the above data set. Finally, we discuss the problems associated with 2-D DIGE gel-based proteomic approaches such as ours and suggest a modified approach for decreased inter-gel variation, improved protein resolution and increased protein concentration, which should significantly facilitate protein identification.     

Identification of distinctive protein expression patterns in colorectal adenoma

Purpose: As a pre‐malignant precursor, adenoma provides an ideal tissue for proteome profiling to investigate early colorectal cancer development and provide possible targets for preventive interventions. The aim of this study was to identify patterns of differential protein expression that distinguish colorectal adenoma from normal tissue. Experimental design: Twenty paired samples of adenoma and normal mucosa were analysed by 2‐DE and MALDI‐TOF/TOF MS to detect proteins with ≥2‐fold differential expression. Results: Four proteins were up‐regulated in adenoma (Annexin A3, S100A11, S100P and eIF5A‐1) and three were down‐regulated (Galectin‐1, S100A9 and FABPL). S100P, galectin‐1, S100A9 and FABPL expression was localised by immunohistochemistry. Conclusions and clinical relevance: Distinctive patterns of in vivo protein expression in colorectal adenoma were identified for the first time. These proteins have important functions in cell differentiation, proliferation and metabolism, and may play a crucial role in early colorectal carcinogenesis. The ability to recognise premalignant lesions may have important applications in cancer prevention.     

Towards a comprehensive proteome of normal and malignant human colon tissue by 2-D-LC-ESI-MS and 2-DE proteomics and identification of S100A12 as potential cancer biomarker

The aim of this study was to characterize the proteome of normal and malignant colonic tissue. We previously studied the colon proteome using 2‐DE and MALDI‐MS and identified 734 proteins (Roeßler, M., Rollinger, W., Palme S., Hagmann, M.‐L., et al.., Clin. Cancer Res. 2005, 11, 6550–6557). Here we report the identification of additional colon proteins from the same set of tissue samples using a complementary nano‐flow 2‐D‐LC‐ESI‐MS. In total, 484 proteins were identified in colon. Of these, 252 had also been identified by the 2‐DE/MALDI‐MS approach, whereas 232 proteins were unique to the 2‐D‐LC‐ESI‐MS analysis. Comparing protein expression in neoplastic and normal colon tissue indicated elevated expression of several proteins in colorectal cancer, among them the well established tumor marker carcinoembryonic antigen, as well as calnexin, 40S ribosomal protein S15a, serpin H1, and S100A12. Overexpression of these proteins was confirmed by immunoblotting. Serum levels of S100A12 were determined by ELISA and were found to be strongly elevated in colorectal cancer patients compared to healthy individuals. We conclude, that 2‐D‐LC‐ESI‐MS is a powerful approach to identify and compare protein profiles of tissue samples, that it is complementary to 2‐DE/MALDI‐MS approaches and has the potential to identify novel biomarkers.     

Differential regulation of redox proteins and chaperones in HbEβ-thalassemia erythrocyte proteome

Purpose: In (hemoglobin, Hb) HbEβ‐thalassemia, HbE (β‐26 Glu→Lys) interacts with β‐thalassemia to produce clinical manifestation of varying severity. This is the first proteomic effort to study changes in protein levels of erythrocytes isolated from HbEβ‐thalassemic patients compared to normal. Experimental design: We have used 2‐DE and MALDI‐MS/MS‐based techniques to investigate the differential proteome profiling of membrane and Hb‐depleted fraction of cytosolic proteins of erythrocytes isolated from the peripheral blood samples of HbEβ‐thalassemia patients and normal volunteers. Results: Our study showed that redox regulators such as peroxiredoxin 2, Cu‐Zn superoxide dismutase and thioredoxin and chaperones such as α‐hemoglobin stabilizing protein and HSP‐70 were upregulated in HbEβ‐thalassemia. We have also observed larger amounts of membrane associated globin chains and indications of disruption of spectrin‐based junctional complex in the membrane skeleton of HbEβ‐thalassemic erythrocytes upon detection of low molecular weight fragments of β‐spectrin and decrease in β‐actin and dematin content. Conclusion and clinical relevance: We have observed interesting changes in the proteomic levels of redox regulators and chaperons in the thalassemic hemolysates and have observed strong correlation or association of the extent of such proteomic changes with HbE levels. This could be important in understanding the role of HbE in disease progression and pathophysiology.     

Reconstructing the pipeline by introducing multiplexed multiple reaction monitoring mass spectrometry for cancer biomarker verification: an NCI-CPTC initiative perspective

Proteomics holds great promise in personalized medicine for cancer in the post-genomic era. In the past decade, clinical proteomics has significantly evolved in terms of technology development, optimization and standardization, as well as in advanced bioinformatics data integration and analysis. Great strides have been made for characterizing a large number of proteins qualitatively and quantitatively in a proteome, including the use of sample fractionation, protein microarrays and MS. It is believed that differential proteomic analysis of high-quality clinical biospecimen (tissue and biofluids) can potentially reveal protein/peptide biomarkers responsible for cancer by means of their altered levels of expression and/or PTMs. Multiple reaction monitoring, a multiplexed platform using stable isotope dilution-MS with sensitivity and reproducibility approaching that of traditional ELISAs commonly used in the clinical setting, has emerged as a potentially promising technique for next-generation high-throughput protein biomarker measurements for diagnostics and therapeutics.     

Proteomics identification of differentially expressed proteins in the muscle of dysferlin myopathy patients

The muscular dystrophies are a large and heterogeneous group of neuromuscular disorders that can be classified according to the mode of inheritance, the clinical phenotype and the molecular defect. To better understand the pathological mechanisms of dysferlin myopathy we compared the protein-expression pattern in the muscle biopsies of six patients with this disease with six patients with limb girdle muscular dystrophy 2A, five with facioscapulohumeral dystrophy and six normal control subjects. To investigate differences in the expression levels of skeletal muscle proteins we used 2-DE and MS. Western blot or immunohistochemistry confirmed relevant results. The study showed specific increase expression of proteins involved in fast-to-slow fiber type conversion (ankyrin repeat protein 2), type I predominance (phosphorylated forms of slow troponin T), sarcomere stabilization (actinin-associated LIM protein), protein ubiquitination (TRIM 72) and skeletal muscle differentiation (Rho-GDP-dissociation inhibitor ly-GDI) in dysferlin myopathy. As anticipated, we also found differential expression of proteins common to all the muscular dystrophies studied. This comparative proteomic analysis suggests that in dysferlin myopathy (i) the type I fiber predominance is an active process of fiber type conversion rather than a selective loss of type II fibers and (ii) the dysregulation of proteins involved in muscle differentiation further confirms the role of dysferlin in this process.     

Wavelet-based procedures for proteomic mass spectrometry data processing

Proteomics aims at determining the structure, function and expression of proteins. High-throughput mass spectrometry (MS) is emerging as a leading technique in the proteomics revolution. Though it can be used to find disease-related protein patterns in mixtures of proteins derived from easily obtained samples, key challenges remain in the processing of proteomic MS data. Multiscale mathematical tools such as wavelets play an important role in signal processing and statistical data analysis. A wavelet-based algorithm for proteomic data processing is developed. A MATLAB implementation of the software package, called WaveSpect0, is presented including processing procedures of step-interval unification, adaptive stationary discrete wavelet denoising, baseline correction using splines, normalization, peak detection, and a newly designed peak alignment method using clustering techniques. Applications to real MS data sets for different cancer research projects in Vanderbilt Ingram Cancer Center show that the algorithm is efficient and satisfactory in MS data mining.     

Decreased annexin A3 expression correlates with tumor progression in papillary thyroid cancer

Purpose : The aim of this study is to identify the potential tumor markers that function in carcinogenesis and tumor progression, thus providing important diagnostic and prognostic information. Experimental design : We performed 2‐D gel electrophoresis and MALDI‐TOF MS to investigate the differentially expressed proteins in 25 papillary thyroid carcinoma tissues. For validation of candidate proteins and investigation of clinical significance, we performed Western, Northern blot analysis and immunohistochemical staining. Results : Our proteomic analyses revealed significantly decreased annexin A3 expression in papillary thyroid carcinoma at both the protein and mRNA levels, compared with normal thyroid tissue. ANXA3 immunoreactivity was not significantly correlated with lymph node metastasis, multifocality, capsular invasion or perithyroidal extension in thyroid cancer. However, the tumor subgroup with a lymph node metastasis score of >3 displayed significantly lower ANXA3 expression than did subgroups with negative and ≤3 scores (p=0.001). Moreover, ANXA3 expression was markedly lower in large tumors (>1 cm in diameter) than in microcarcinomas (p=0.001). Conclusion and clinical relevance : Decreased expression of ANXA3 in papillary thyroid cancer supports the idea that ANXA3 may be an effective marker of microcarcinoma, and a negative predictor of papillary thyroid cancer progression.     

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.     

Comparative proteomic analysis of differentially expressed proteins in an in vitro cellular carcinogenesis model of oral squamous cell carcinoma

In vitro cellular model is an important tool to be used to investigate the cellular events related to pathophysiological conditions in humans. We have developed an in vitro cellular carcinogenesis model of oral squamous cell carcinoma (OSCC). In this study, we performed comparative proteomic analysis using 2‐DE and LC‐tandem mass chromatography to separate and identify differentially expressed proteins. Forty‐five proteins were identified, including 24 proteins with decreased expression and 19 proteins with increased expression during carcinogenesis from immortalized oral epithelial cells to squamous cancerous cells. The identified known proteins were classified into three ontologies of cellular component, molecular function, and biological process. Further validation of five identified proteins (ANXA1, ANXA2, CTSB, KRT17, and S100A6) in the cellular carcinogenesis model and cancerous tissues from OSCC patients confirmed the comparative proteomic results. Moreover, Annexin A1 and A2 expression levels correlated with the pathological differentiation grade of cancerous tissues. Thus, this work provides a dynamic protein file of differentially expressed proteins in oral squamous carcinoma cells, which could provide clues to study the mechanisms of OSCC carcinogenesis and possibly be developed as potential biomarkers for clinical diagnosis or prognostic monitoring.     

Impact of incomplete DNase I treatment on human macrophage proteome analysis

The aim of our study was to analyze the proteomic pattern of human macrophages obtained over a 4 year period from blood donors. The purpose was to simulate a long-term clinical study to assess the application of 2-D DIGE technique for differential proteomic analysis of these scarce samples. Bioinformatic analysis of 2-D DIGE gels of 19 different cultures of macrophages assessed whether they did or did not contain at least specific five spots identified by MS as being or containing bovine deoxyribonuclease I (DNase I). Bovine DNase I was used during sample treatment to remove nucleic acids from protein extracts. Macrophages were classified in two groups, which appeared to be differentiated by the completeness of DNase I treatment. Further detailed analysis revealed a different proteomic pattern of macrophage protein samples according to the completeness of this treatment. The major group of proteins affected, accounting for one third of the differentially expressed proteins, included proteins involved in cell motion and actin cytoskeleton reorganization. The use of DNase I for the removal of nucleic acids from protein samples must be avoided in proteomic studies since it can generate bias in the analysis of protein expression patterns.     

Identification of glial-cell-line-derived neurotrophic factor-regulated proteins of striatum in mouse model of Parkinson disease

Glial-cell-line-derived neurotrophic factor (GDNF) is a potent survival factor for dopaminergic neurons, and hence serves as a therapeutic candidate for the treatment of Parkinson's disease. However, despite the potential clinical and physiological importance of GDNF, its mechanism of action is unclear. Therefore, we employed a state-of-the-art proteomic technique, DIGE, along with MS and a bioinformatics tool called Database for Annotation, Visualization and Integrated Discovery (DAVID), to profile proteome changes in the parkinsonian mouse striatum after GDNF challenge. Forty-six unique differentially expressed proteins were successfully identified, which were found either up-regulated and/or down-regulated at the two time points 4 and 72 h compared with the control. Proteins involved in cell differentiation and system development formed the largest part of the proteins regulated under GDNF. Furthermore, the aberrant expression of HSPs and mitochondria-associated proteins were noticeable. Moreover, mitochondrial stress 70 protein and heat shock cognate 71 kDa protein, whose relative levels increased significantly in GDNF-treated striatum, were further evaluated with Western blot and RT-PCR, demonstrating a good agreement with quantitative proteomic data. These data will provide some clues for understanding the mechanisms by which GDNF promotes the survival of dopaminergic neurons.     

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.     

Functional protease profiling for diagnosis of malignant disease

Clinical proteomic profiling by mass spectrometry (MS) aims at uncovering specific alterations within mass profiles of clinical specimens that are of diagnostic value for the detection and classification of various diseases including cancer. However, despite substantial progress in the field, the clinical proteomic profiling approaches have not matured into routine diagnostic applications so far. Their limitations are mainly related to high-abundance proteins and their complex processing by a multitude of endogenous proteases thus making rigorous standardization difficult. MS is biased towards the detection of low-molecular-weight peptides. Specifically, in serum specimens, the particular fragments of proteolytically degraded proteins are amenable to MS analysis. Proteases are known to be involved in tumour progression and tumour-specific proteases are released into the blood stream presumably as a result of invasive progression and metastasis. Thus, the determination of protease activity in clinical specimens from patients with malignant disease can offer diagnostic and also therapeutic options. The identification of specific substrates for tumour proteases in complex biological samples is challenging, but proteomic screens for proteases/substrate interactions are currently experiencing impressive progress. Such proteomic screens include peptide-based libraries, differential isotope labelling in combination with MS, quantitative degradomic analysis of proteolytically generated neo-N-termini, monitoring the degradation of exogenous reporter peptides with MS, and activity-based protein profiling. In the present article, we summarize and discuss the current status of proteomic techniques to identify tumour-specific protease-substrate interactions for functional protease profiling. Thereby, we focus on the potential diagnostic use of the respective approaches.     

Proteomic analysis of pericardial effusion: Characteristics of tuberculosis-related proteins

The aim of this study has been designed to identify the tuberculosis (TB)-related proteins in pericardial effusion by proteomic approaches. TB is one of the major infectious diseases causing pericardial effusion. This study details protein profiles in pericardial effusion from three TB patients and three heart failure patients. Pericardial effusions were analyzed using 2-DE combined with the nano-HPLC-ESI-MS/MS. Eleven protein spots with differential expression in pericardial effusion were identified between the two groups of TB and heart failure patients (the control group). Seven protein spots were upregulated and four were downregulated. The composition of the pericardial effusion proteome may reflect the pathophysiological conditions affecting the progression of tuberculous pericarditis. The proteins in the tuberculous pericardial effusion with differential expression may serve as new and direct indicators of drug treatment. A possible conclusion is indicated that fibrinogen may play an important role for fibrin assembly in tuberculous pericardial effusion.     

Colorectal cancer proteomics, molecular characterization and biomarker discovery

Colorectal cancer (CRC) is a widespread disease, whose major genetic changes and mutations have been well characterized in the sporadic form. Much less is known at the protein and proteome level. Still, CRC has been the subject of multiple proteomic studies due to the urgent necessity of finding clinically relevant markers and to elucidate the molecular mechanisms underlying the progression of the disease. These proteomic approaches have been limited by different technical issues, mainly related with sensitivity and reproducibility. However, recent advances in proteomic techniques and MS systems have rekindled the quest for new biomarkers in CRC and an improved molecular characterization. In this review, we will discuss the application of different proteomic approaches to the identification of differentially expressed proteins in CRC. In particular, we will make a critical assessment about the use of 2-D DIGE, MS and protein microarray technologies, in their different formats, to identify up- or downregulated proteins and/or autoantibodies profiles that could be useful for CRC characterization and diagnosis. Despite a wide list of potential biomarkers, it is clear that more scientific efforts and technical advances are still needed to cover the range of low-abundant proteins, which may play a key role in CRC diagnostics and progression.     

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.     

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.