Cover Picture: Proteomics – Clinical Applications 1/2008

In this issue of Proteomics you will find the following highlighted articles: Colon Cancer Complements to 2‐DE from 2‐D‐LC “When you have a good thing going, run with it” – Quote from paleolithic philosopher and hunting consultant. In this case, Thierolf et al. took a colon cancer sample set well‐characterized by 2‐DE‐MALDI PMF and ran it through a 2‐D‐LC‐ESI‐MS protocol. The samples of malignant and normal tissues from the same patient, analyzed by 2‐DE‐MALDI and mass fingerprinting (reported elsewhere) yielded 734 unique proteins. When the same tissue specimens were analyzed by 2‐D‐LC‐ESI‐MS, 484 proteins were identified, 232 of them new and 252 that had been ID’d in the earlier work. The two unique sets exhibited similar functional and ontological profiles, confirming the complementarity of the two methods. Using the data to select up‐regulated proteins for evaluation of potential for serving as biomarkers, they chose S100A12 as particularly interesting. An ELISA found that it was more sensitive but less discriminating than carcinoembryonic antigen. S100A12 is also an inflammation marker. Thierolf, M. et al., Proteomics Clin. Appl. 2008, 2, 11–22 Urinary bladder cancer: A proteomic approach Standing at number five on the US cancer frequency list, urinary bladder cancer costs almost $3 billion a year. No acceptable early detection tests have been developed – it seems no one will volunteer for a “routine” annual cystoscopy, so the 5‐year survival rate has stayed below 50% for many years. Several urinary biomarkers have been developed but fall short in their frequency of false positives or false negatives. Using 2‐DE/MALDI‐TOF MS and Ingenuity Systems’ Pathway Analysis software, Li et al. surveyed invasive urothelial carcinomas for up‐regulated proteins and settled on Choro­ideremia‐like protein (CHML) out of 21 candidates. Immunohistochemistry and Western blotting verified the specificity. The functional pathways found are part of the lipid metabolism, inflammation and molecular transport machinery and CHML is part of the Rab geranylgeranylation pathway. More work is needed to understand the diagnostic potential of CHML. Li, J. et al., Proteomics Clin. Appl. 2008, 2, 78–89. Angina: Looking for markers in all the right places Angina, the chest pain associated with heart attacks, has two forms: stable (SAP) and unstable (UAP). SAP is relieved by rest. UAP pain persists at rest and is often due to formation of a clot which can lead to major or fatal damage (ischemia, myocardial infarct). The ability to distinguish the two would be a boon to hospital emergency care facilities because admission and intensive care are not required for SAP. Brown et al. report here the use of anti‐leukocyte antibody arrays to analyze circulating cells by surface CD antigen type. Starting with 82 antibodies, they could readily distinguish healthy from SAP and UAP cases from 8 and 19 spot intensity differences, respectively. SAP and UAP could be separated with seven markers using spot intensity and cluster analysis, but not as cleanly, possibly because SAP has a tendency to convert to UAP. Brown, A. et al., Proteomics Clin. Appl. 2008, 2, 90–98.     

In this issue: Proteomics – Clinical Applications 1/2008

In this issue of Proteomics you will find the following highlighted articles: Colon Cancer Complements to 2‐DE from 2‐D‐LC “When you have a good thing going, run with it” – Quote from paleolithic philosopher and hunting consultant. In this case, Thierolf et al. took a colon cancer sample set well‐characterized by 2‐DE‐MALDI PMF and ran it through a 2‐D‐LC‐ESI‐MS protocol. The samples of malignant and normal tissues from the same patient, analyzed by 2‐DE‐MALDI and mass fingerprinting (reported elsewhere) yielded 734 unique proteins. When the same tissue specimens were analyzed by 2‐D‐LC‐ESI‐MS, 484 proteins were identified, 232 of them new and 252 that had been ID’d in the earlier work. The two unique sets exhibited similar functional and ontological profiles, confirming the complementarity of the two methods. Using the data to select up‐regulated proteins for evaluation of potential for serving as biomarkers, they chose S100A12 as particularly interesting. An ELISA found that it was more sensitive but less discriminating than carcinoembryonic antigen. S100A12 is also an inflammation marker. Thierolf, M. et al., Proteomics Clin. Appl. 2008, 2, 11–22 Urinary bladder cancer: A proteomic approach Standing at number five on the US cancer frequency list, urinary bladder cancer costs almost $3 billion a year. No acceptable early detection tests have been developed – it seems no one will volunteer for a “routine” annual cystoscopy, so the 5‐year survival rate has stayed below 50% for many years. Several urinary biomarkers have been developed but fall short in their frequency of false positives or false negatives. Using 2‐DE/MALDI‐TOF MS and Ingenuity Systems’ Pathway Analysis software, Li et al. surveyed invasive urothelial carcinomas for up‐regulated proteins and settled on Choro­ideremia‐like protein (CHML) out of 21 candidates. Immunohistochemistry and Western blotting verified the specificity. The functional pathways found are part of the lipid metabolism, inflammation and molecular transport machinery and CHML is part of the Rab geranylgeranylation pathway. More work is needed to understand the diagnostic potential of CHML. Li, J. et al., Proteomics Clin. Appl. 2008, 2, 78–89. Angina: Looking for markers in all the right places Angina, the chest pain associated with heart attacks, has two forms: stable (SAP) and unstable (UAP). SAP is relieved by rest. UAP pain persists at rest and is often due to formation of a clot which can lead to major or fatal damage (ischemia, myocardial infarct). The ability to distinguish the two would be a boon to hospital emergency care facilities because admission and intensive care are not required for SAP. Brown et al. report here the use of anti‐leukocyte antibody arrays to analyze circulating cells by surface CD antigen type. Starting with 82 antibodies, they could readily distinguish healthy from SAP and UAP cases from 8 and 19 spot intensity differences, respectively. SAP and UAP could be separated with seven markers using spot intensity and cluster analysis, but not as cleanly, possibly because SAP has a tendency to convert to UAP. Brown, A. et al., Proteomics Clin. Appl. 2008, 2, 90–98.     

Overexpression and elevated plasma level of tumor-associated antigen 90K/Mac-2 binding protein in colorectal carcinoma

The cancer cell secretome may contain potentially useful biomarkers. Previously, we have analyzed the colorectal carcinoma (CRC) cell secretome. In this study, tumor‐associated antigen 90K (TAA90K)/Mac‐2 binding protein (Mac‐2BP), one of the CRC cell secreted proteins, was chosen for evaluation as a potential CRC biomarker because its mRNA level was also found to be significantly elevated in CRC tissues and in a more metastatic CRC cell line from the analysis of two public domain array‐based datasets. Immunohistochemical analysis of 241 CRC specimens showed that TAA90K/Mac‐2BP was positively detected in 52.7% of the tumors, but weakly or not detected in over 95% of the adjacent nontumor epithelial cells. The plasma TAA90K/Mac‐2BP levels were significantly higher in CRC patients (N = 280) versus healthy controls (N = 147) (7.77 ± 3.49 vs. 5.72 ± 2.67 μg/mL, p<0.001). Moreover, combination of TAA90K/Mac‐2BP and carcinoembryonic antigen (CEA) could outperform CEA alone in discriminating CRC patients from healthy persons in this case‐control study. Our results collectively indicate that analysis of cancer cell secretome is a feasible strategy for identifying cancer biomarker candidates, and the TAA90K/Mac‐2BP may be a potential CRC biomarker.     

Clinical applications of capillary electrophoresis coupled to mass spectrometry in biomarker discovery: Focus on bladder cancer

A major requirement in the application of proteins as clinical biomarkers is that they provide a highly sensitive and specific result in disease assessment. Since single biomarkers are generally of limited accuracy, a group or panel of well-characterized biomarkers appears appropriate, providing a more robust and sensitive MS-based analytical platform. CE coupled to MS has been successfully used in biomarker discovery and application, as it enables the selective detection of peptides and small proteins, combining the high separation capacity of CE with the advanced sensitivity of MS. CE-MS allows the characterization of highly complex samples (such as urine, plasma, and other biofluids) in a consistent and reproducible way. It has a range of applications, many focusing especially in studies on urinary peptide biomarkers in kidney and cardiovascular diseases. Another major field of interest has been malignancy of the genitourinary system. In the first part of this review, we cover technical aspects and performance characteristics of CE-MS, with special focus on the requirements for biomarker discovery and clinical application. In the second part, we review the potential and development of CE-MS in the management of genitourinary cancers, especially bladder cancer. CE-MS has been employed in several studies aimed at discovering biomarkers for bladder cancer that may be useful in diagnosis, monitoring for recurrence, and prediction of the risk for the muscle-invasive stage. In the last part of the review, we discuss current challenges and provide an outlook for ongoing and possible future developments.     

Cover Picture: Proteomics – Clinical Applications 12/2008

In this issue of Proteomics – Clinical Applications you will find the following highlighted articles: Looking through the leftovers The magic in conditioned medium has been recognized for decades but exactly what it was and how it worked has only begun to come to light more recently. Byproducts of metabolic activity are easy to spot, more challenging are the secreted growth factors and other types of communication molecules. Ogura et al. looked at the litter that surrounded various cancer cells as a potential gold mine. It took only a bit of RP‐HPLC and MALDI‐TOF panning to turn up nuggets of pro‐ and pre‐proneurotensin/neuromedin N (pre‐proNT/NTN), good candidates for small‐cell lung cancer biomarkers. Pre‐proNT/NTN was found in medium from 4 out of 7 different small‐cell lung carcinomas but 0 out of 8 non‐small‐cell carcinomas. Ogura, S.‐i. et al., Proteomics Clin. Appl. 2008, 2, 1620–1627. Hunting for Huntington's Creating animal models of inherited human diseases is not always a simple issue of replacing an animal gene with the human equivalent. Huntington's Disease (HD), caused at least in part by the development of expanded polyglutamine (CAG)n sequences in the huntingtin gene, has been modeled in mice with n>60 (CAG)n sequences but this leads to expression as a juvenile form of the disease. Nguyen et al. have developed transgenic rats that come much closer to the pattern of adult human HD in the character and time of appearance of motor deficits, cognitive decline, and emotional disturbance. After thorough micro array evaluation of the rat model at ages 3 and 12 months, these researchers argue that they are much closer to a system suitable for selection and application of biomarkers and potential therapeutics. Nguyen, H. P. et al., Proteomics Clin. Appl. 2008, 2, 1638–1650. Bifunctional assay means less work To be able to have your cake and eat it, too, is one of those things economists tell us is impossible. Don't tell Rader et al. though. They are investigating methods to simplify and speed up human papilloma virus (HPV) biomarker screening from the same sample. This would be very useful for cervical cancer staging, still a problem despite the recent introduction and growing use of a vaccine for the most frequent cancer‐causing HPV types. Cervical samples were collected into a tube containing an RNA stabilizing reagent, from which proteins could be extracted and freed of cervical mucus. RNA could be extracted from the same samples with Trizol reagent according to the manufacturer's directions. Proteins recovered were cleanly analyzed by 2‐D DIGE and Western blots; total RNA could be analyzed on human cDNA arrays. Rader, J. S. et al., Proteomics Clin. Appl. 2008, 2, 1658–1669.     

Detection of colorectal adenoma and cancer based on transthyretin and C3a-desArg serum levels

Colorectal cancer is the second leading cause of cancer death, and it develops from benign colorectal adenomas in over 95% of patients. Early detection of these cancer precursors by screening tests and their removal can potentially eradicate more than 95% of colorectal cancers before they develop. To discover sensitive and specific biomarkers for improvement of pre‐clinical diagnosis of colorectal adenoma and cancer, we analysed in two independent studies (n = 87 and n = 83 patients) serum samples from colorectal cancer (stage III), colorectal adenoma and control patients using SELDI‐TOF‐MS. Extensive statistical analysis was performed to establish homogeneous patient groups based on their clinical data. Two biomarkers that were each able to distinguish control patients from either colorectal adenoma or colorectal cancer patients (p<0.001) were identified as transthyretin (pre‐albumin) and C3a‐desArg by MS/MS and were further validated by antibody‐based assays (radial immunodiffusion, ELISA). A combination of both proteins clearly indicated the presence of colorectal adenoma or carcinoma. Using a cut‐off of <0.225 g/L for transthyretin and >1974 ng/mL for C3a‐desArg, we found a sensitivity and specificity for colorectal adenoma of 96% and 70%, respectively.     

In this issue: Proteomics – Clinical Applications 12/2008

In this issue of Proteomics – Clinical Applications you will find the following highlighted articles: Looking through the leftovers The magic in conditioned medium has been recognized for decades but exactly what it was and how it worked has only begun to come to light more recently. Byproducts of metabolic activity are easy to spot, more challenging are the secreted growth factors and other types of communication molecules. Ogura et al. looked at the litter that surrounded various cancer cells as a potential gold mine. It took only a bit of RP‐HPLC and MALDI‐TOF panning to turn up nuggets of pro‐ and pre‐proneurotensin/neuromedin N (pre‐proNT/NTN), good candidates for small‐cell lung cancer biomarkers. Pre‐proNT/NTN was found in medium from 4 out of 7 different small‐cell lung carcinomas but 0 out of 8 non‐small‐cell carcinomas. Ogura, S.‐i. et al., Proteomics Clin. Appl. 2008, 2, 1620–1627. Hunting for Huntington's Creating animal models of inherited human diseases is not always a simple issue of replacing an animal gene with the human equivalent. Huntington's Disease (HD), caused at least in part by the development of expanded polyglutamine (CAG)n sequences in the huntingtin gene, has been modeled in mice with n>60 (CAG)n sequences but this leads to expression as a juvenile form of the disease. Nguyen et al. have developed transgenic rats that come much closer to the pattern of adult human HD in the character and time of appearance of motor deficits, cognitive decline, and emotional disturbance. After thorough micro array evaluation of the rat model at ages 3 and 12 months, these researchers argue that they are much closer to a system suitable for selection and application of biomarkers and potential therapeutics. Nguyen, H. P. et al., Proteomics Clin. Appl. 2008, 2, 1638–1650. Bifunctional assay means less work To be able to have your cake and eat it, too, is one of those things economists tell us is impossible. Don't tell Rader et al. though. They are investigating methods to simplify and speed up human papilloma virus (HPV) biomarker screening from the same sample. This would be very useful for cervical cancer staging, still a problem despite the recent introduction and growing use of a vaccine for the most frequent cancer‐causing HPV types. Cervical samples were collected into a tube containing an RNA stabilizing reagent, from which proteins could be extracted and freed of cervical mucus. RNA could be extracted from the same samples with Trizol reagent according to the manufacturer's directions. Proteins recovered were cleanly analyzed by 2‐D DIGE and Western blots; total RNA could be analyzed on human cDNA arrays. Rader, J. S. et al., Proteomics Clin. Appl. 2008, 2, 1658–1669.     

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.     

Comparison of proteomic biomarker panels in urine and serum for ovarian cancer diagnosis

Purpose : The purposes of this study were to confirm previously found candidate epithelial ovarian cancer biomarkers in urine and to compare a paired serum biomarker panel and a urine biomarker panel from the same study cohort with regard to the receiver operating characteristic curve (ROC) area under the ROC curve (AUC) values. Experimental design : Four significant urine biomarkers were confirmed among 130 pelvic mass patients in the present study. The four biomarkers form a potential urine biomarker panel. From the same study cohort, the potential urine biomarker panel was compared to a serum biomarker panel, consisting of seven proteins/peptides, OvaRI. Results : Multivariate analysis of the urine panel demonstrated a significant differentiation (p<0.0001) between epithelial ovarian cancer patients and patients with benign ovarian pelvic masses. The ROC AUC of the urine panel was 0.84 and the ROC AUC of OvaRI was 0.83. Combining the urine panel with OvaRI demonstrated a significant contribution from both, for urine peaks, OR=2.12 and for OvaRI, OR=1.39; the ROC AUC of this model was 0.88. Conclusions and clinical relevance : We demonstrated that both urine and serum can be used individually or in combination to potentially aid in ovarian cancer diagnostics. Urine proteomic profiling could provide biomarkers for the non‐invasive test required in clinical practice.     

Considerations for powering a clinical proteomics study: Normal variability in the human plasma proteome

Proteomics is increasingly being applied to the human plasma proteome to identify biomarkers of disease for use in non‐invasive assays. 2‐D DIGE, simultaneously analysing thousands of protein spots quantitatively and maintaining protein isoform information, is one technique adopted. Sufficient numbers of samples must be analysed to achieve statistical power; however, few reported studies have analysed inherent variability in the plasma proteome by 2‐D DIGE to allow power calculations. This study analysed plasma from 60 healthy volunteers by 2‐D DIGE. Two samples were taken, 7 days apart, allowing estimation of sensitivity of detection of differences in spot intensity between two groups using either a longitudinal (paired) or non‐paired design. Parameters for differences were: two‐fold normalised volume change, α of 0.05 and power of 0.8. Using groups of 20 samples, alterations in 1742 spots could be detected with longitudinal sampling, and in 1206 between non‐paired groups. Interbatch gel variability was small relative to the detection parameters, indicating robustness and reproducibility of 2‐D DIGE for analysing large sample sets. In summary, 20 samples can allow detection of a large number of proteomic alterations by 2‐D DIGE in human plasma, the sensitivity of detecting differences was greatly improved by longitudinal sampling and the technology was robust across batches.     

Membrane Proteome of Invasive Retinoblastoma: Differential Proteins and Biomarkers

Purpose: Retinoblastoma (RB) is a pediatric ocular cancer which is caused due to the aberrations in the RB1 gene. The changes in the membrane proteomics would help in understanding the development of the retinoblastoma and could identify candidates for biomarkers and therapy. Experimental design: Quantitative proteomics is performed on the enriched membrane fractions from pooled normal retina (n = 5) and pooled retinoblastoma tissues (n = 5). The proteins are tryptic‐digested and tagged with iTRAQ labels. Orbitrap mass spectrometry is used to analyze and quantify the deregulated membrane proteins involved in the RB tumor progression. Immunohistochemistry (IHC) is used to further validate few of the differentially expressed proteins. Results: A total of 3122 proteins are identified of which, 663 proteins are found to be deregulated with ≥two fold change in the RB tumor compared to the retina. 282 proteins are upregulated and 381 are downregulated with ≥2 peptide identifications. Bioinformatic analysis revealed that, most of the proteins are involved in the transport, cellular communication, and growth. Overexpression of lamin B1 (LMNB1) and transferrin receptor (TFRC) are observed in RB tumors using IHC. Conclusion and clinical relevance: The present study, is the first comprehensive quantitative membrane proteomic atlas of the differentially regulated proteins in RB compared to the retina. LMNB1 and TFRC could be potential biomarkers for this childhood cancer.     

Discovery of putative oocyte quality markers by comparative ExacTag proteomics

Purpose: Identification of the biomarkers of oocyte quality, and developmental and reprogramming potential is of importance to assisted reproductive technology in humans and animals. Experimental design: PerkinElmer ExacTag? Kit was used to label differentially proteins in pig oocyte extracts (oocyte proteome) and pig oocyte‐conditioned in vitro maturation media (oocyte secretome) obtained with high‐ and low‐quality oocytes. Results: We identified 16 major proteins in the oocyte proteome that were expressed differentially in high‐ versus low‐quality oocytes. More abundant proteins in the high‐quality oocyte proteome included kelch‐like ECH‐associated protein 1 (an adaptor for ubiquitin‐ligase CUL3), nuclear export factor CRM1 and ataxia‐telangiectasia mutated protein kinase. Dystrophin (DMD) was more abundant in low‐quality oocytes. In the secretome, we identified 110 proteins, including DMD and cystic fibrosis transmembrane conductance regulator, two proteins implicated in muscular dystrophy and cystic fibrosis, respectively. Monoubiquitin was identified in the low‐quality‐oocyte secretome. Conclusions and clinical implications: A direct, quantitative proteomic analysis of small oocyte protein samples can identify potential markers of oocyte quality without the need for a large amount of total protein. This approach will be applied to discovery of non‐invasive biomarkers of oocyte quality in assisted human reproduction and in large animal embryo transfer programs.     

Observation of peptide differences between cancer and control in gastric juice

Biomarkers for various diseases have been extensively searched for the past 5 years. Nevertheless, most efforts were focused on the search for protein biomarkers from serum samples. In this work, we tried to look for peptide biomarkers from gastric juice samples with MALDI‐TOF‐MS. More than 200 gastric juice samples from healthy people, gastric ulcer patients, duodenal ulcer patients, and cancer patients were examined. There were clear pattern differences of mass spectra among samples from healthy people and patients with different gastric diseases. We found five peptides for gastric cancer diagnosis with high sensitivity and specificity. Sequences of these five peptides, including two pepsinogen fragments, leucine zipper protein fragment, albumin fragment, and α‐1‐antitrypsin fragment, have been identified by mass spectrometric analysis and immuno‐deplete assay with antibodies.     

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.     

New horizon for breast cancer biomarker discoveries: What might the liquid biopsy of nipple aspirate fluid hold?

The existence of cellular, molecular and biochemical heterogeneity of human breast cancers reveals the intricacy of biomarkers complexity, stimulating studies on new approaches (like “liquid biopsies”) for the improvements in precision medicine. Breast cancer is recognized as a leading cause of morbidity and mortality worldwide with tumors significantly diverse and containing many types of cells showing different genetic and epigenetic profiles. In this field, the technology of liquid biopsy (applied to a fluid produced by breast gland, named nipple aspirate fluids, NAF) highlights the power of combining basic and clinical research. NAF is the mirror of the entire ductal/alveolar breast tree providing almost complete proteomic profile and a valuable source for biomarker discovery, in non‐invasive manner than tissue biopsies. The liquid biopsy technology using NAF may represent the outstanding breakthrough of proteomic cancer research revealing novel diagnostic and prognostic applications. In conjunction to metabolomic and degradome profiling, the use of NAF as liquid biopsy approach will improve the detection of changes in the cellular microenvironment of the breast tumors, understanding molecular and biochemical mechanisms which drive breast tumor initiation, maintenance and progression, and finally enhancing the development of novel drug targets and new treatment strategies.     

Cancer biomarker discovery via low molecular weight serum proteome profiling – Where is the tumor?

Time‐course analyses of rapidly processed serum performed in parallel by SELDI and nanoscale LC‐MS/MS have revealed the temporal correlation of several literature‐based disease markers with ex vivo driven events such that their in vivo existence in healthy subjects is questionable. Identification by MS/MS reveals these putative biomarkers to be byproducts of the coagulation cascade and platelet activation and suggests plasmatic analysis may be preferred. In a pilot plasmatic study, a cohort of naïve prostate cancer (PCa) samples were uniformly distinguished from their age‐matched controls (n = 20) on the basis of multiple peptidic components; most notably by a derivative of complement C₄ at 1863 m/z (GLEEELQFSLGSKINVK, C4_1353–1369). The fully tryptic nature of this and other putative PCa discriminants is consistent with the cleavage specificity of common blood proteases and questions the need for tumor‐derived proteolytic activities as has been proposed. In light of the known correlation of disregulated hemostasis with malignant disease, we suggest the underlying differentiating phenomena in these types of analyses may lie in the temporal disparity of sample activation such that the case (patient) samples are preactivated while the control samples are not.     

An immunoproteomic approach for identification of clinical biomarkers of Whipple's disease

Whipple's disease (WD) is a chronic multisystemic infection, caused by the bacterium Tropheryma whipplei. The main clinical presentations are classic WD (CWD) with histologic lesions in the gastrointestinal tract, endocarditis, and isolated neurologic infection. The current strategy for diagnosis remains invasive.The present study aimed to select the protein candidates for serological diagnosis of WD. The first step was to identify candidate proteins by an immunoproteomic approach combining 2‐DE using a total extract of a T. whipplei, immunoblotting, and MS. The second step was to validate the discovered biomarkers using a recombinant protein‐based ELISA. Serum samples from 18 patients with WD and from 54 control individuals were tested. A sugar ABC transporter, TWT328 (sensitivity (Se) 61%, specificity (Sp) 87%, positive predictive value (PPV) 61%, negative predictive value (NPV) 87%, and positive likelihood ratio (PLR) 4.69) was the best marker for development of serodiagnosis for CWD. We also obtained a reproducible immunoreactive protein pattern for patients with isolated neurological infection due to T. whipplei (Se 100%, Sp 93%, PPV 55.5%, NPV 100%, and PLR 13.51) as an encouraging step towards noninvasive diagnosis of this particular manifestation. Nine recombinant candidates have been successfully screened with serum samples. Results from these ELISA assays skewed with those obtained with immunoblots.     

Proteomic analysis for human urinary proteins associated with arsenic intoxication

Arsenic is widely distributed in nature and is mainly transported in the environment by water. Consumption of artesian well water with high levels of arsenic has been associated with genitourinary cancer, especially bladder transitional cell carcinoma (TCC). To search for biomarkers that are specific for arsenic associated with the diagnosis of bladder TCC, proteins in the urine of non-cancer urological patients and patients with either bladder TCC or arsenic-associated bladder TCC were systematically examined by HPLC ESI-MS/MS. Urine specimens were collected by catheterization from patients and age- (within 5?years) and sex-matched non-cancer urological patients. A nano-HPLC-ESI-MS/MS was used to generate proteome patterns from urine specimens obtained from patients with arsenic-associated (n?=?8) and non-arsenic-associated (n?=?8) primary TCC and from sex- and age-matched non-cancer urological patients (n?=?8). Three urinary proteins were found to have significantly altered levels in patients following chronic arsenic exposure. These proteins were a disintegrin and metalloprotease (ADAM) protein, a calpain9, and ring finger protein 20. The large-scale identification of urinary proteomes using HPLC ESI-MS/MS may serve as an ideal and efficient method to establish a panel of potential arsenic-associated TCC biomarkers and may help elucidate the mechanisms involved in bladder cancer induced by chronic arsenic exposure.     

Proteomic analysis of sera of asymptomatic,early‐stage patients with Wilson's disease

Wilson's disease (WD) is characterized by excessive accumulation of intracellular copper in liver and extrahepatic tissues, leading to significant oxidative stress and tissue damage. To date, several diagnostic biomarkers for WD such as serum ceruloplasmin, serum or urine copper levels and copper content in liver have been identified. However, these biomarkers may not be convincing for the diagnosis in some WD patients. To identify additional novel diagnostic biomarkers, we compared the serum protein profiles of asymptomatic childhood WD patients (n=20), without neurologic manifestation or liver cirrhosis, with normal controls (n=13). Fourteen spots, five up‐regulated and nine down‐regulated (>2‐fold), were differentially expressed in WD patients in comparison to normal control on 2‐DE. Among them, three spots were down‐regulated in both male and female WD. MS/MS analysis revealed that the three spots were complement component C3, complement factor B and alpha‐2 macroglobulin. By comparative proteome analysis, complement component C3, complement factor B and alpha‐2 macroglobulin, which are related to oxidative stress and inflammation, turned out to be good candidates for novel diagnostic biomarkers for early stages of WD.     

Developing proteomics-based biomarkers for colorectal neoplasms for clinical practice: Opportunities and challenges

Colorectal cancer (CRC) arises from the normal colon epithelium through the accumulation of genetic mutations and epigenetic alterations that are associated with progression along the histological adenoma-adenocarcinoma sequence. Elucidating the molecular alterations underlying disease progression will not only provide insight into the behavior of the tumors, but also could lead to the discovery of useful biomarkers for diagnosis, monitoring treatment responsiveness, or predicting disease outcomes. In the past a few years, there have been several evaluating differentially expressed protein biomarkers by employing proteomics technologies coupled with mass spectrometry. In the current review, we will briefly summarize the results from selected recent studies using tissue or serum samples from CRC patients in the past 5 years and discuss the opportunities and challenges in translating these findings from the research setting to clinical practice.