Identification of Apolipoprotein AI as a serum biomarker of chronic kidney disease in liver transplant recipients, using proteomic techniques

Chronic kidney disease (CKD) is a common complication post‐orthotopic liver transplantation (OLT). Development of CKD is detected by monitoring serum urea and creatinine, however disease can occasionally be at an advanced stage before they become abnormal. Therefore, more accurate parameters are required. In order to identify novel biomarkers of CKD, serum was obtained from 47 OLT recipients with CKD (glomerular filtration rate <60 mL/min) and 23 with normal renal function (glomerular filtration rate >90 mL/min). Using the proteomic technique SELDI‐TOF‐MS, three protein biomarkers (55.6 kDa, 9.5 kDa and 11.4 kDa) were identified that, together, could stratify patients into cases or controls with a sensitivity and specificity of 93.6 and 91.3%, respectively. The area under the curve was 0.94. The primary splitter of the groups at 55.6 kDa was an alternative version of a molecule at 27.8 kDa, which was subsequently identified by 1‐D SDS‐PAGE and LC‐ESI‐MS/MS to be Apolipoprotein AI. Protein expression was shown to be reduced in CKD, by both ELISA (p = 0.057) and Western blot analysis (p = 0.003). Apolipoprotein AI is a novel, accurate marker of CKD post‐OLT. It does require further validation in a large, more diverse patient population but could potentially improve detection of CKD.     

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.     

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.     

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.     

Profiling the potential biomarkers for cell differentiation of pancreatic cancer using iTRAQ and 2-D LC-MS/MS

Pancreatic cancer is a highly lethal disease that is difficult to diagnose at early stage and even more difficult to cure. SW1990 and PANC-1 represent the two cancer cell lines, which are both derived from pancreatic duct, but at different cell differentiation stages. In this study, we applied the iTRAQ-labeling technology and 2-D strong cation exchange/reversed phase liquid chromatography – LC-MS/MS) to profile the secreted proteins of SW1990 and PANC-1 cells in a conditioned cell culture medium. A total of 401 proteins were identified by MS/MS and protein database searching, the percentages of these proteins predicted in the categories of plasma membrane, intracellular and secreted proteins were 29.2, 32.7 and 38.2%, respectively. Fifty six proteins were identified with unknown functions and 19 proteins were quantified with significant level changes between the two cancer cell lines under the specific cell condition with 12 proteins being up-regulated (>1.3-fold change) in PANC-1 (e.g. FLJ31222 protein, 97 kDa protein, type IV collagenase precursor, 38 kDa protein and centaurin) and seven proteins being up-regulated in SW1990 (e.g. fibroblast growth factor receptor substrate 2, putative p150, hypothetical protein LOC 654463 and LOC 55701). The proteins with significant level changes may provide a baseline to investigate mechanisms underlying the differentiation of two cell lines and can be further screened for better protein biomarkers in pancreatic cancer.     

Identification of circulating endorepellin LG3 fragment: Potential use as a serological biomarker for breast cancer

Comparative proteome analysis was performed on the cultured media of human nontumor and malignant breast cell lines, Hs578Bst and Hs578T, respectively, in search of a serological biomarker(s) for breast cancer. Proteins in the conditioned media were separated by 2‐D PAGE and then visualized by silver‐staining. Eight proteins changed differentially by more than two‐fold were identified by MALDI‐TOF/TOF MS. Among the proteins identified, the terminal laminin‐like globular (LG3) domain of endorepellin, which was recently reported as an antiangiogenesis factor, was decreased in the cancer cell line. We confirmed the bone morphogenic protein‐1 (BMP‐1) mediated cleavage site on the N‐terminus of endorepellin LG3 fragment. This finding suggests that the LG3 fragment is specifically released by a BMP‐1 driven limited proteolytic process. The protein was also detected in plasma by Western blot analysis and selected reaction monitoring (SRM). The plasma level of the endorepellin LG3 fragment was significantly lower in breast cancer patients compared to healthy donors (p = 0.017; n = 12). The LG3 protein concentration in the control plasma was measured at approximately 3.7 pmol/mL compared to 1.8 pmol/mL in plasma from the cancer patients. We suggest that these results support the potential use of the endorepellin LG3 fragment as a new serological biomarker for breast cancer.     

Proteomic characterization of differentially expressed proteins in breast cancer: Expression of hnRNP H1, RKIP and GRP78 is strongly associated with HER-2/neu status

The human epidermal growth factor receptor type 2 (HER‐2/neu) oncoprotein is overexpressed in about 30% of breast cancers and associates with metastatic phenotypes of breast tumours. Dissecting the HER‐2/neu‐modulated molecules in cancer will be helpful in elucidating the underlying molecular mechanisms of HER‐2/neu‐driven tumourigenesis. We investigated the differential proteome profiles between microdissected HER‐2/neu‐positive and ‐negative tumours and unambiguously identified 21 proteins with diverse biological functions by peptide sequencing and NCBInr database interrogation. Six proteins were up‐regulated whereas 15 were down‐regulated in the HER‐2/neu‐positive tumours. Differential expressions of heterogeneous nuclear ribonucleoprotein H1 (hnRNP H1), 78 kDa glucose‐regulated protein (GRP78/Bip) and Raf‐1 kinase inhibitor protein (RKIP), which have not been previously reported as being linked to HER‐2/neu signalling, were further verified. Immunohistochemical staining on tissue microarray sections demonstrated a positive correlation of hnRNP H1 (p = 0.008) and negative correlations of GRP78 and RKIP (p = 0.018 and 0.013, respectively) with HER‐2/neu. Heregulin α1 enhanced hnRNP H1, but reduced GRP78 and RKIP expression in BT474 cells in a dose‐dependent manner, providing evidence of crosstalk between HER‐2/neu signalling and these modulators. Our studies have identified novel modulators that are likely to be intricately involved in HER‐2/neu‐driven tumour proliferation, invasion and metastasis.     

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.     

Translating N‐Glycan Analytical Applications into Clinical Strategies for Ovarian Cancer

Protein glycosylation, particularly N‐linked glycosylation, is a complex posttranslational modification (PTM), which plays an important role in protein folding and conformation, regulating protein stability and activity, cell–cell interaction, and cell signaling pathways. This review focuses on analytical techniques, primarily MS‐based techniques, to qualitatively and quantitatively assess N‐glycosylation while successfully characterizing compositional, structural, and linkage features with high specificity and sensitivity. The analytical techniques explored in this review include LC–ESI–MS/MS and MALDI time‐of‐flight MS (MALDI‐TOF‐MS), which have been used to analyze clinical samples, such as serum, plasma, ascites, and tissue. Targeting the aberrant N‐glycosylation patterns observed in MALDI–MS imaging (MSI) offers a platform to visualize N‐glycans in tissue‐specific regions. The studies on the intra‐patient (i.e., a comparison of tissue‐specific regions from the same patient) and inter‐patient (i.e., a comparison of tissue‐specific regions between different patients) variation of early‐ and late‐stage ovarian cancer (OC) patients identify specific N‐glycan differences that improve understanding of the tumor microenvironment and potentially improve therapeutic strategies for the clinic.     

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.     

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.     

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.     

Shotgun immunoproteomics to identify disease‐associated bacterial antigens: Application to human colon cancer

Circulating antibodies reflect a mirror view of invading antigens that are related to infection and cancer. This was recently exemplified by using serum antibodies to capture Streptococcus bovis antigens followed by MS to generate antigen profiles that were diagnostic for colon cancer. These bacterial antigen profiles have a high potential to aid in the immuno‐diagnosis of this disease, as the magnitude of the immune response to bacterial antigens is, in general, superior to the immune response against tumor (self) antigens. In this study, the identity of individual colon cancer‐associated streptococcal antigens was revealed by enrichment of these “diagnostic” antigens by selected patient antibodies followed by high‐accuracy nanoLC‐MS/MS peptide identification. This showed that both the histone‐like protein HlpA and the ribosomal protein Rp L7/L12 are members of the colon cancer‐associated S. bovis immunome. Both antigens also seem to belong to the group of anchorless surface proteins, like 14 additional proteins that were co‐identified in S. bovis cell wall extracts. Among these were the known streptococcal anchorless surface proteins GAPDH and Enolase. Taken together, these data show that shotgun immunoproteomics, combining immunocapture in‐line with LC MS/MS, is a convenient approach for the rapid identification of disease‐associated bacterial antigens.     

Cell‐Free DNA Blood Collection Tubes Are Appropriate for Clinical Proteomics: A Demonstration in Colorectal Cancer

Background

Optimized blood collection tubes (BCT) have been developed to expand the utility of plasma cell‐free DNA (cfDNA) and are in clinical use. The appropriateness of plasma collected and stored in these tubes for proteomic analysis is unknown.

Methods

Paired blood samples were collected in BCT and traditional K3EDTA (EDTA) tubes from healthy controls and from colorectal cancer (CRC) patients before and after surgery, and stored for between 45 min and 48 h at room temperature. Plasma proteins were analyzed following high‐abundant plasma protein depletion in quantitative discovery and targeted proteomics by liquid chromatography tandem‐mass spectrometry (LC‐MS/MS).

Results

BCT reduced cellular protein contamination in healthy controls over time, and increased the number of high confident low‐abundant protein identifications in CRC blood samples compared to matched samples collected in EDTA tubes. The known CRC plasma protein biomarker, carcinoembryonic antigen (CEA), showed elevated levels across patients pre‐operatively when collected and stored in BCT compared to EDTA tubes. Emerging CRC biomarkers, Dickkopf‐3 (DKK3) and Gelsolin (GSN), showed elevated levels pre‐operatively when collected in BCT.

Conclusions

Optimized BCT are appropriate for low‐abundant plasma protein analysis and can be used with confidence for clinical proteomics.

     

Evaluation of a type 1 diabetes serum cohort by SELDI-TOF MS protein profiling

Proteomics analysis of serum from patients with type 1 diabetes (T1D) may lead to novel biomarkers for prediction of disease and for patient monitoring. However, the serum proteome is highly sensitive to sample processing and before proteomics biomarker research serum cohorts should preferably be examined for potential bias between sample groups. SELDI‐TOF MS protein profiling was used for preliminary evaluation of a biological‐bank with 766 serum samples from 270 patients with T1D, collected at 18 different paediatric centers representing 15 countries in Europe and Japan over 2 years (2000–2002). Samples collected 1 (n = 270), 6 (n = 248), and 12 (n = 248) months after T1D diagnosis were grouped across centers and compared. The serum protein profiles varied with collection site and day of analysis; however, markers of sample processing were not systematically different between samples collected at different times after diagnosis. Three members of the apolipoprotein family increased with time in patient serum collected 1, 6, and 12 months after diagnosis (ANOVA, p<0.001). These results support the use of this serum cohort for further proteomic studies and illustrate the potential of high‐throughput MALDI/SELDI‐TOF MS protein profiling for evaluation of serum cohorts before proteomics biomarker research.     

Implementation of defected ground structure for microstrip filtenna design

A microstrip patch filtenna inspired by defected ground structure (DGS) is presented in this article. It uses modified split ring resonator and capacitance loaded strip as a radiating element. The presented structure is incorporated with a pair of double U‐shaped DGS (DU‐DGS) to obtain filtering characteristics. The width of DU‐DGS plays a vital role in selecting attenuation poles of the filter as well as for the filtenna circuit. The separation distance between the DU‐DGS also affects the resonant frequency of the structure. Both radiation and filtration can be performed through a single structure, otherwise known as filtenna. The physical size of the proposed filtenna in terms of guided wavelength is 2.465λ_g × 1.160λ_g × 0.116λ_g at 10.8 GHz, and is comparatively less to others reported, so is considered as a superior feature. The presented filtenna possesses impedance bandwidth of 700 and 1800 MHz at 10.8 and 16.6 GHz, which covers standards of X‐ and Ku‐band, respectively. So, this can be referred to as dual band filtenna. The radiation pattern shows omnidirectionality in both E and H planes at resonance.     

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.