Serological proteome analysis to identify systemic candidiasis patients in the intensive care unit: Analytical, diagnostic and prognostic validation of anti-Candida enolase antibodies on quantitative clinical platforms

Systemic candidiasis (SC) is associated with high morbidity and mortality, because it generally affects patients with severe underlying diseases and its diagnosis is difficult and often delayed, resulting in delayed therapy. We used serological proteome analysis to screen serum anti‐Candida IgG antibody‐reactivity profiles in 24 patients under intensive care, 12 of which had confirmed SC (fungal cultures), and in 12 healthy subjects. A total of 15 immunogenic proteins from Candida albicans protoplast lysates were differentially immunorecognized by serum IgG antibodies from SC patients compared to controls. Two‐way hierarchical clustering and principal‐component analyses of these antibody‐reactivity patterns accurately differentiated SC patients from controls. Anti‐Eno1p IgG antibodies were found to be present at high abundance in SC patients and be an important molecular fingerprint in serum for SC diagnosis. Differential anti‐Eno1p IgG antibody reactivity was further validated by a tag capture ELISA and a Western blot assay in 45 SC patients and 118 non‐SC subjects. Both quantitative assays provided comparable analytical, diagnostic and prognostic performances, and verified initial proteomic‐profiling results. If confirmed in prospective cohort studies, these anti‐Eno1p IgG antibodies might be useful for SC diagnosis. However, these, at least as measured by these clinical platforms, appear to have limited prognostic value in SC patients.     

Changes of serum‐associated fucosylated glycoproteins and changes in glycosylation of IgA in human cirrhosis

Many modifications in N‐glycosylation have been demonstrated in hepatic cirrhosis. These modifications correspond to an increase of a bisecting core alpha(1,6)‐fucosylated biantennary glycan, an increase in core fucosylation, and the presence of an important population of neutral oligosaccharides in human serum of cirrhotic patients. In this study, a glycoproteomic approach which consists of lectin affinity chromatography, MALDI‐TOF MS for the characterization of N‐glycans released from glycoproteins, one‐ and 2‐D PAGE, electrospray ionization quadrupole ion trap (ESI‐QIT) MS was used to identify serum fucosylated glycoproteins related to cirrhosis. Employing this method, we have shown that IgA is one of the major proteins that is responsible of the glycosylation modifications observed in the serum N‐glycome of cirrhotic patients. To our knowledge, this is the first time that aberrant N‐glycosylation of IgA in cirrhosis is described.     

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.     

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.     

Proteomics integrated with Escherichia coli vector-based vaccines and antigen microarrays reveals the immunogenicity of a surface sialidase-like protein of Propionibacterium acnes

Proteomics is a powerful tool for the identification of proteins, which provides a basis for rational vaccine design. However, it is still a highly technical and time‐consuming task to examine a protein's immunogenicity utilizing traditional approaches. Here, we present a platform for effectively evaluating protein immunogenicity and antibody detection. A tetanus toxin C fragment (Tet‐c) was used as a representative antigen to establish this platform. A cell wall‐anchoring sialidase‐like protein (SLP) of Propionibacterium acnes was utilized to assess the efficacy of this platform. We constructed an Escherichia coli vector‐based vaccine by overexpressing Tet‐c or SLP in E. coli and utilized an intact particle of E. coli itself as a vaccine (E. coli Tet‐c or SLP vector). After ultraviolet (UV) irradiation, the E. coli vector‐based vaccines were administered intranasally into imprinting control region mice without adding exogenous adjuvants. For antibody detection, we fabricated antigen microarrays by printing with purified recombinant proteins including Tet‐c and SLP. Our results demonstrated that detectable antibodies were elicited in mice 6 weeks after intranasal administration of UV‐irradiated E. coli vector‐based vaccines. The antibody production of Tet‐c and SLP was significantly elevated after boosting. Notably, the platform with main benefits of using E. coli itself as a vaccine carrier provides a critical template for applied proteomics aimed at screening novel vaccine targets. In addition, the novel immunogenic SLP potentially serves as an antigen candidate for the development of vaccines targeting P. acnes‐associated diseases.     

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.     

Whole cell profiling and identification of galectin-1 as a potential marker of renal cell carcinoma

For most cancers, the patient's prognosis improves dramatically if the disease is detected at an early stage. Although advancements in imaging technology have improved early detection, many cancers remain undetected until it is too late for curative intervention. We have established, for the first time, expression difference mapping analysis of whole cell proteins from renal cell carcinoma (RCC) cell lines using ProteinChip technology. A total of 20 different RCC cell lines were cultured in vitro directly on ProteinChip arrays for 24 h. Direct MS analysis of proteins from the attached cells showed identical protein profiles by all analysed RCC lines. Comparative on‐chip analysis of isolated malignant cells from native tumour specimens revealed protein patterns highly similar to those from the continuous RCC lines. However, cultured primary cortex cells showed specific protein differences. Differential protein profiling of isolated cytosolic and enriched membrane fractions from the RCC lines revealed that the protein pattern of the membrane proteins included or were identical to those of the entire cells. Proteomics analysis of the chip‐binding membrane fractions allowed the identification of three forms of galectin‐1 as potential RCC marker. ProteinChip analysis with a bound‐specific antibody certified that galectin‐1 could be an RCC marker. Immunostaining methods confirmed the overexpression of galectin‐1 in renal carcinoma in comparison to healthy tissue.     

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.     

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.     

Cover Picture: Proteomics – Clinical Applications 9/2008

In this issue of Proteomics – Clinical Applications you will find the following highlighted articles: Always probing for more: prostate biomarkers It feels a bit like the late nineteenth century, but instead of a gold rush every two to five years, it's a new favorite target in the biomarker rushes. (Actually, gold rushes go back to ancient Egypt. Biomarkers don't go that far but medical research does.) Here, Burgess et al. take a walk outside the box when they encounter the asymmetry of protein abundance. Rather than synthetically trapping compounds to expose or capture low abundance compounds, they use nature's own: in particular, alpha‐2‐macroglobulin (A2M). A2Ms normal function is to bind proteins that are to be protected from proteolysis, a universal protease inhibitor. Using immunoprecipitation of A2M and comparing cases vs. controls, enhanced levels of heat shock protein 90 in serum was their most interesting candidate for this year's marker rush. Burgess, E. F. et al., Proteomics Clin. Appl. 2008, 2, 1223–1233. Brainwashing samples No, we are not suggesting 1984‐style re‐education to improve proteome productivity. Rather, Dean et al. are reporting on the efficiency of fractionation of brain tissue proteins by graduated detergent extraction prior to 2‐DE. Another anticipated benefit is increased relative concentration of the less abundant proteins. Samples from two areas of the human brain (Brodmann's Area 9 (BA9) and caudate nucleus and putamen CP) were prepared with a sequential extraction kit and compared by 1‐DE and Western blots, 2‐DE and MALDI‐TOF. The conclusion was that no detergent conditions were found that resolved proteins completely but that each detergent point gave a different 2‐D pattern, a benefit for those looking for distinguishing marks. Dean, B. et al., Proteomics Clin. Appl. 2008, 2, 1281–1289. Liver and kidney pie In orthotopic (“full replacement”) liver transplants, one of the most common complications is chronic kidney (yes, kidney) disease. Currently, kidney complications are tracked by functional tests, like serum urea and creatinine levels. If things look suspicious, glomerular filtration rates can be checked. O'Riordan et al. applied SELDI TOF‐MS techniques to serum samples to look for easier, more accurate targets. Serum samples were collected repeatedly over a 6‐month period. Each was divided into six fractions by elution pH or by organic solvent, then examined on weak cation exchange (CM10), hydrophobic (H50) and immobilized metal affinity surfaces (IMAC30). CM10 was best at distinguishing case from control using three proteins and reporting a sensitivity of ～87–94%. On the basis of peptide LC‐MS and 1‐D SDS‐PAGE and confirmed by ELISA, the best single indicator was APO‐AI. Most cases of kidney disease appeared to be linked to the use of calcineurin inhibitors for immune suppression. O'Riordan, A. et al., Proteomics Clin. Appl. 2008, 2, 1338–1348.     

Two‐dimensional gel electrophoresis of apolipoprotein C‐III and other serum glycoproteins for the combined screening of human congenital disorders of O‐ and N‐glycosylation

Congenital disorders of glycosylation (CDG) are inherited diseases that can affect not only the N‐glycan (e.g. CDG type I and II) but also the O‐glycan biosynthesis pathway. In the absence of specific clinical symptoms, there is a need for a reliable biological screening of these two groups of CDG. Using a few microlitres of human serum, 2‐DE and immunoblotting were applied to the separation and simultaneous detection of the isoforms of the O‐glycosylated protein apolipoprotein C‐III (apoC‐III) and of four N‐glycosylated proteins, namely alpha‐antitrypsin, alpha‐1 acid glycoprotein, haptoglobin and transferrin. For the study of O‐glycosylation, this technique allowed the reliable separation of the three fractions of apoC‐III and the determination of normal percentage values in an adult population. Concerning N‐glycosylation, the study of serum samples from patients with CDG type Ia revealed marked abnormalities systematically affecting the four 2‐DE separated N‐linked glycoproteins. 2‐DE coupled to immunoblotting using a mixture of specific antibodies could be easily and reliably employed for the combined screening of both N‐ and O‐glycosylation disorders in humans.     

In this issue: Proteomics – Clinical Applications 9/2008

In this issue of Proteomics – Clinical Applications you will find the following highlighted articles: Always probing for more: prostate biomarkers It feels a bit like the late nineteenth century, but instead of a gold rush every two to five years, it's a new favorite target in the biomarker rushes. (Actually, gold rushes go back to ancient Egypt. Biomarkers don't go that far but medical research does.) Here, Burgess et al. take a walk outside the box when they encounter the asymmetry of protein abundance. Rather than synthetically trapping compounds to expose or capture low abundance compounds, they use nature's own: in particular, alpha‐2‐macroglobulin (A2M). A2Ms normal function is to bind proteins that are to be protected from proteolysis, a universal protease inhibitor. Using immunoprecipitation of A2M and comparing cases vs. controls, enhanced levels of heat shock protein 90 in serum was their most interesting candidate for this year's marker rush. Burgess, E. F. et al., Proteomics Clin. Appl. 2008, 2, 1223–1233. Brainwashing samples No, we are not suggesting 1984‐style re‐education to improve proteome productivity. Rather, Dean et al. are reporting on the efficiency of fractionation of brain tissue proteins by graduated detergent extraction prior to 2‐DE. Another anticipated benefit is increased relative concentration of the less abundant proteins. Samples from two areas of the human brain (Brodmann's Area 9 (BA9) and caudate nucleus and putamen CP) were prepared with a sequential extraction kit and compared by 1‐DE and Western blots, 2‐DE and MALDI‐TOF. The conclusion was that no detergent conditions were found that resolved proteins completely but that each detergent point gave a different 2‐D pattern, a benefit for those looking for distinguishing marks. Dean, B. et al., Proteomics Clin. Appl. 2008, 2, 1281–1289. Liver and kidney pie In orthotopic (“full replacement”) liver transplants, one of the most common complications is chronic kidney (yes, kidney) disease. Currently, kidney complications are tracked by functional tests, like serum urea and creatinine levels. If things look suspicious, glomerular filtration rates can be checked. O'Riordan et al. applied SELDI TOF‐MS techniques to serum samples to look for easier, more accurate targets. Serum samples were collected repeatedly over a 6‐month period. Each was divided into six fractions by elution pH or by organic solvent, then examined on weak cation exchange (CM10), hydrophobic (H50) and immobilized metal affinity surfaces (IMAC30). CM10 was best at distinguishing case from control using three proteins and reporting a sensitivity of ～87–94%. On the basis of peptide LC‐MS and 1‐D SDS‐PAGE and confirmed by ELISA, the best single indicator was APO‐AI. Most cases of kidney disease appeared to be linked to the use of calcineurin inhibitors for immune suppression. O'Riordan, A. et al., Proteomics Clin. Appl. 2008, 2, 1338–1348.     

Investigation of an albumin‐enriched fraction of human serum and its albuminome

The removal of albumin and other high abundance proteins is a routine first step in the analysis of serum and plasma proteomes. However, as albumin can bind proteins and peptides, there is a universal concern as to how the serum proteome is changed by the removal of albumin. To address this concern, the current study was designed to identify proteins and peptides removed from the serum during albumin depletion; to determine which of these are bound to albumin (rather than copurified) and whether the bound proteins are intact proteins or peptide fragments. Sequential, independent analyses including both anti‐albumin antibody (anti‐HSA) affinity chromatography and SEC were used to isolate albumin‐bound proteins. RP‐HPLC and 1‐D SDS‐PAGE were then used to further separate the proteins prior to identification by MS/MS. Finally, whole protein molecular weight (MW) MS measurements coupled with protein coverage obtained by MS were combined to assess whether the bound proteins were intact or peptide fragments. Combining the results from multiple approaches, 35 proteins, of which 24 are intact, were found to be associated with albumin, and they include both known high and low abundance proteins.     

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.     

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.     

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.     

Two dimensional gel electrophoresis of apolipoprotein C‐III and MALDI‐TOF MS are complementary techniques for the study of combined defects in N‐ and mucin type O‐glycan biosynthesis

In the field of diseases related to glycosylation disorders, congenital defects associated with abnormalities in both O‐ and N‐glycosylation of proteins constitute arising novel entities. Defects in subunits of the conserved oligomeric Golgi protein complex have been shown to be involved in an important part of previously unsolved CDG type II combining abnormalities in both mucin type core1 O‐ and N‐glycans; furthermore, recent studies revealed that autosomal recessive cutis laxa type II could also be associated with such combined glycosylation defects. Based on the studies of serum samples from three patients including a case of cutis laxa, we present here evidence that 2‐DE of apolipoprotein C‐III in combination with MALDI‐TOF‐MS analysis of serum O‐ and N‐glycans allow the detection and the biochemical characterization of these newly recognized glycosylation disorders.     

Identification of melanoma‐specific serological markers using proteomic analyses

The discovery of novel melanoma markers for not only early detection but also monitoring disease status is promising to improve the clinical outcome of patients. In the present study, we performed proteomic comparative analysis of plasma proteins between healthy volunteers and melanoma patients using NanoLC and MALDI‐TOF‐MS. As a result, we were successful in identifying nine proteins that were specifically expressed in melanoma plasma compared with healthy plasma, most of which had not previously been identified as plasma markers of melanoma. The mRNA expression levels of four proteins [pro‐platelet basic protein precursor (PPBP), serum amyloid A2 (SAA2), complement factor H‐related protein 1 precursor (FHR1), inter‐alpha‐trypsin inhibitor heavy chain H4 precursor (IAIH4)] were prominently up‐regulated in several melanoma cell lines compared with melanocytes. Moreover, two proteins (PPBP, SAA) were shown to be expressed in tumor specimens from melanoma patients. In the survival time analysis regarding melanoma patients, the semi‐quantified plasma PPBP levels were statistically negatively correlated with the survival time. Most interestingly, the significant survival benefit was seen in low PBPP level group (< index 20) versus high level (≥ index 20) group. The results suggest that PPBP might be a novel promising serological marker and a prognostic factor specific to melanomas.     

Post-transplant proteinuria associated with everolimus: Definition of main features with proteomics

Little is known on both the composition and mechanism(s) of proteinuria associated with the use of mTOR inhibitors, in particular of Everolimus (E). We characterized urinary proteins utilizing an integrated proteomics approach (quantitative essays, 2‐DE, MALDI‐TOF, Western blot) in 48 renal transplant recipients who were alternatively treated with E (n = 31) or with enteric coated mycophenolic acid (EC‐MPA) (n = 17). Twelve E patients (39%) developed high (>3 g/day) or intermediate proteinuria (1–3 g) compared to four (23%) of the EC‐MPA group. Urinary proteins (p<0.001), β2 microglobulin (p<0.001) and α1microglobulin (p<0.025) were higher in E than in EC‐MPA, appeared more rapidly and were inversely correlated with the day of treatment. Proteomics showed a marked increase of all urinary components in E and EC‐MPA patients, major changes involving typical components of glomerular damage (albumin, α1‐Zn glycoprotein, α2HS glycoprotein, leucin‐richα2‐glycoprotein) and specific bio‐markers for E (clusters of α1‐antitrypsin fragments and monoclonal λ chains). Finally, inter‐α‐trypsin‐inhibitor heavy chain H4 precursor was decreased in E and EC‐MPA urine compared to normal urine. In conclusion, E induced massive and generalized proteinuria of mixed glomerular and tubular origin that was correlated with the start of treatment and reached a nephrotic range in few cases. Specific urinary markers reflect renal alterations related to the transplant or specific alterations associated with the drug.