Automated serum peptide profiling using novel magnetic C18 beads off-line coupled to MALDI-TOF-MS

Serum peptide profiling by MS is an emerging approach for disease diagnosis and biomarker discovery. A magnetic bead‐based method for off‐line serum peptide capture coupled to MALDI‐TOF‐MS has been recently introduced. However, the reagents are not available to the general scientific community. Here, we developed a protocol for serum peptide capture using novel magnetic C18 beads, and automated the procedure on a high‐throughput magnetic particle processor. We investigated bead equilibration, peptide binding and peptide elution conditions. The method is evaluated in terms of peaks counts and reproducibility of ion intensities in control serum. Overall, the DynaBead‐RPC18‐based serum sample processing protocol reported here is reproducible, robust and allows for the detection of ?200 peptides at m/z 800–4000 of serum that was allowed to clot for 1 h. The average intra‐experiment %CV of normalized ion intensities for crude serum and 0.5% TFA/0.15% n‐octyl glucoside‐treated serum, respectively, were 12% (range 2–38%) and 10% (3–21%) and the inter‐experiment %CVs were 24% (10–53%) and 31% (10–59%). Importantly, this method can be used for serum peptide profiling by anyone in possession of a MALDI‐TOF instrument. In conjunction with the KingFisher® 96, the whole serum peptide capture procedure is high‐throughput (?20 min per isolation of 96 samples in parallel), thereby facilitating large‐scale disease profiling studies.     

Protein profiling in pathology: analysis and evaluation of 239 frozen tissue biopsies for diagnosis of B-cell lymphomas

Purpose : We determined the potential value of protein profiling of tissue samples by assessing how precise this approach enables discrimination of B‐cell lymphoma from reactive lymph nodes, and how well the profiles can be used for lymphoma classification. Experimental design : Protein lysates from lymph nodes (n=239) from patients with the diagnosis of reactive hyperplasia (n=44), follicular lymphoma (n=63), diffuse large B‐cell lymphoma (n=43), mantle cell lymphoma (n=47), and chronic lymphocytic leukemia/small lymphocytic B‐cell lymphoma (n=42) were analysed by SELDI‐TOF MS. Data analysis was performed by (i) classification and regression tree‐based analysis and (ii) binary and polytomous logistic regression analysis. Results : After internal validation by the leave‐one‐out principle, both the classification and regression tree and logistic regression classification correctly identified the majority of the malignant (87 and 96%, respectively) and benign cases (73 and 75%, respectively). Classification was less successful since approximately one‐third of the cases of each group were misclassified according to the histological classification. However, an additional mantle cell lymphoma case that was misclassified as chronic lymphocytic leukemia/small lymphocytic B‐cell lymphoma initially was identified based on the protein profile. Conclusions and clinical relevance : SELDI‐TOF MS protein profiling allows for reliable identification of the majority of malignant lymphoma cases; however, further validation and testing robustness in a diagnostic setting is needed.     

Assessment by Matrix‐Assisted Laser Desorption/Ionization Time‐of‐Flight Mass Spectrometry of the Effects of Preanalytical Variables on Serum Peptidome Profiles Following Long‐Term Sample Storage

Purpose

Human serum and plasma are often used as clinical specimens in proteomics analyses, and peptidome profiling of human serum is a promising tool for identifying novel disease‐associated biomarkers. Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) is widely used for peptidomic biomarker discovery. Careful sample collection and handling are required as either can have a profound impact on serum peptidome patterns, yet the effects of preanalytical variables on serum peptidome profiles have not been completely elucidated. The present study investigated the effects of preanalytical variables, including storage temperature, duration (up to 12 months), and thawing methods, on MALDI‐TOF MS‐based serum peptidome patterns.

Experimental design

Aliquots of serum samples were pretreated with weak cation exchanger magnetic beads using an automated ClinProtRobot system and then analyzed by MALDI‐TOF MS.

Results

A number of significant differences in peak intensities were observed depending on sample processing variables.

Conclusions and clinical relevance

These peaks can be used as sample quality markers to assess the effects of long‐term storage on serum peptidome profiles using MALDI‐TOF MS.

     

Acute phase serum amyloid A in ovarian cancer as an important component of proteome diagnostic profiling

In the context of serum amyloid A (SAA) identification as ovarian cancer marker derived by SELDI‐MS, its serum levels were measured by immunoassay in different stages of ovarian cancer, in benign gynecological tumors, and in healthy controls. In addition, SELDI‐TOF‐MS spectra were obtained by protocol optimized for the SAA peak intensity. SELDI data on small proteins (5.5–17.5 kDa) and SAA immunoassay data were combined with cancer antigen (CA)125 data in order to study the classification accuracy between cancer and noncancer by support vector machine (SVM), logistic regression, and top scoring pair classifiers. Although an addition of SAA immunoassay data to CA125 data did not significantly improve cancer/noncancer discrimination, SVM applied to combined biomarker data (CA125 and SAA immunoassay variables plus 48 SELDI peak variables) yielded the best classification rate (accuracy 95.2% vs. 86.2% for CA125 alone). Notably, most of discriminatory peaks selected by the classifiers have significant correlation with the major known peaks of SAA (11.7 kDa) and transthyretin (13.9 kDa). Acute phase serum amyloid A (A‐SAA) was proved to be an important member of cancer discriminatory protein profile. Among the eight known ovarian cancer SELDI profile components, A‐SAA is the most relevant to molecular pathogenesis of cancer and it has the highest degree of up‐regulation in disease.     

Salivary biomarkers associated with perceived satiety and body mass in humans

Regulation of food intake and energy homeostasis is controlled by a delicate balancing of numerous central and peripheral factors, including circulating peptide hormones. This study investigated the proteome of saliva using SELDI‐TOF‐MS in relation to satiety and body mass index (BMI) in humans. Within a longitudinal test session, 18 subjects were exposed to a lunch‐induced hunger‐satiety shift, while every 15 min collecting their whole saliva and rating their hunger and satiety. Saliva was analysed by SELDI‐TOF‐MS using IMAC arrays with a chromatographic copper surface (IMAC‐Cu). From all subjects and time points measured, peptide and protein profiles showed 190 common peaks. Their interrelationships show that 37% of the variation was accounted for in one dimension. About 30 means had a strong association (0.70<|r|<0.95) with all subjective satiety ratings across time during the test session, and seven peaks were significantly correlated to BMI. Database MS searches indicated characterisation of some relevant metabolic peptide hormones. In conclusion, SELDI‐TOF‐MS on human saliva provides a valuable and noninvasive way of profiling that enables characterisation of novel and differently expressed peptides and proteins which can be used as biomarkers of satiety and overweight.     

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.     

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.     

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.     

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.     

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.     

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

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

In this issue: Proteomics – Clinical Applications 4/2008

In this issue of Proteomics you will find the following highlighted articles: MALDI and methyl groups take on lysosomal storage disease diagnosis Lysosomal storage disorders (LSD) result from the absence or loss of any of a wide variety of glycan‐processing enzymes that lead to accumulation of incompletely processed glyco‐molecules in lysosomes. Diagnosis of the particular type of LSD requires identification of the accumulated species, usually from urine. LSD diagnosis has been a target for many years, including use of GC/MS, TLC, NMR, HPLC, FACE and other techniques. Faid et al. take a simple approach that is quite successful and much less time consuming. After permethylating crude urine, a sample is first run on GC/MS to look for free sialic acid, an indicator of one set of diseases, then a cleaned up sample (C_1–8) is analyzed by MALDI/TOF to characterize glycoproteins and glycopeptides present. The glycans were further analyzed by sequential exoglucosidase digestion on the MALDI target. Sulfated glycocompound analysis requires a desulfation step prior to permethylation. Faid, V. et al., Proteomics Clin. Appl. 2008, 2, 528–542. Popular prostate problem receives piercing look Prostate cancer (PCa) is one of the most frequent male cancers. It is also one of the most frequently misdiagnosed cancers. Prostate specific antigen (PSA) has improved the accuracy somewhat but still generates 700 000 false positives requiring biopsies per year in the US. And very low PSA levels don’t assure freedom from PCa. In short, there is no good biomarker for PCa. Theodorescu et al. recognized that anything recovered from blood had a good chance of being at least partially degraded so they looked at first void urine as a more likely source of stable material. Applying capillary electrophoresis coupled to TOF‐MS, the authors developed an “informative” panel of eight peptides that distinguished between first void and mid‐stream samples and a PCa‐specific panel of 12 proteins that detected the disease. Incorporating the 12 proteins with age and free PSA, sensitivity was 91%, specificity 69%. Theodorescu, D. et al., Proteomics Clin. Appl. 2008, 2, 556–570. No candy in candidiasis Candida albicans is a unicellular yeast that is the third most common nosocomial agent in ICU’s, costing ～US$ 40 000 per incident. Early diagnosis of systemic candidiasis (SC) is crucial for successful control of the infection by antifungal therapies. Blood cultures are not fast and tissue biopsies are not sensitive but are the current “gold standards.” Pitarch et al. report here a serum protein marker for SC: anti‐Candida enolase (Eno1p) IgG. Comparing healthy with SC patients for antibodies against Candida cytoplasmic antigens, 15 antigens were recognized by the SC patients. The strongest response was to Eno1p which was the only antigen to produce a response in all 12 patients. The IgG was tested for its suitability as an antigen in Western and capture ELISA tests. Although the tests appear reliable for detecting SC, they were not good for prognosis. A considerable amount of work remains for full qualification of the test. Pitarch, A. et al., Proteomics Clin. Appl. 2008, 2, 596–618.     

Cover Picture: Proteomics – Clinical Applications 4/2008

In this issue of Proteomics you will find the following highlighted articles: MALDI and methyl groups take on lysosomal storage disease diagnosis Lysosomal storage disorders (LSD) result from the absence or loss of any of a wide variety of glycan‐processing enzymes that lead to accumulation of incompletely processed glyco‐molecules in lysosomes. Diagnosis of the particular type of LSD requires identification of the accumulated species, usually from urine. LSD diagnosis has been a target for many years, including use of GC/MS, TLC, NMR, HPLC, FACE and other techniques. Faid et al. take a simple approach that is quite successful and much less time consuming. After permethylating crude urine, a sample is first run on GC/MS to look for free sialic acid, an indicator of one set of diseases, then a cleaned up sample (C_1–8) is analyzed by MALDI/TOF to characterize glycoproteins and glycopeptides present. The glycans were further analyzed by sequential exoglucosidase digestion on the MALDI target. Sulfated glycocompound analysis requires a desulfation step prior to permethylation. Faid, V. et al., Proteomics Clin. Appl. 2008, 2, 528–542. Popular prostate problem receives piercing look Prostate cancer (PCa) is one of the most frequent male cancers. It is also one of the most frequently misdiagnosed cancers. Prostate specific antigen (PSA) has improved the accuracy somewhat but still generates 700 000 false positives requiring biopsies per year in the US. And very low PSA levels don’t assure freedom from PCa. In short, there is no good biomarker for PCa. Theodorescu et al. recognized that anything recovered from blood had a good chance of being at least partially degraded so they looked at first void urine as a more likely source of stable material. Applying capillary electrophoresis coupled to TOF‐MS, the authors developed an “informative” panel of eight peptides that distinguished between first void and mid‐stream samples and a PCa‐specific panel of 12 proteins that detected the disease. Incorporating the 12 proteins with age and free PSA, sensitivity was 91%, specificity 69%. Theodorescu, D. et al., Proteomics Clin. Appl. 2008, 2, 556–570. No candy in candidiasis Candida albicans is a unicellular yeast that is the third most common nosocomial agent in ICU’s, costing ～US$ 40 000 per incident. Early diagnosis of systemic candidiasis (SC) is crucial for successful control of the infection by antifungal therapies. Blood cultures are not fast and tissue biopsies are not sensitive but are the current “gold standards.” Pitarch et al. report here a serum protein marker for SC: anti‐Candida enolase (Eno1p) IgG. Comparing healthy with SC patients for antibodies against Candida cytoplasmic antigens, 15 antigens were recognized by the SC patients. The strongest response was to Eno1p which was the only antigen to produce a response in all 12 patients. The IgG was tested for its suitability as an antigen in Western and capture ELISA tests. Although the tests appear reliable for detecting SC, they were not good for prognosis. A considerable amount of work remains for full qualification of the test. Pitarch, A. et al., Proteomics Clin. Appl. 2008, 2, 596–618.     

Biomarker discovery in glomerular diseases using urinary proteomics

Renal biopsy remains the gold standard test for definitive diagnosis of glomerular diseases. This invasive procedure; however, has a potential risk for serious complications and is contraindicated in some patients. It is therefore essential to search for noninvasive biomarkers for the diagnosis and prognosis of glomerular diseases. The urine is the most appropriate sample for biomarker discovery in glomerular diseases. Urinary proteomics has thus gained a wide acceptance and has been extensively applied to this area. This review focuses mainly on applications of proteomic technologies to urinary proteome profiling for biomarker discovery in various glomerular diseases, including diabetic nephropathy, IgA nephropathy, membranous nephropathy, focal segmental glomerulosclerosis, primary membranoproliferative glomerulonephritis, lupus nephritis, antiglomerular basement membrane disease, minimal change disease, and pediatric nephrotic syndrome. Recent findings from these studies are summarized and discussed. These data clearly underline the great promise of urinary proteomics in biomarker discovery for glomerular diseases.     

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.     

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.     

Potential biomarkers for oral squamous cell carcinoma: Proteomics discovery and clinical validation

Oral squamous cell carcinoma (OSCC) is the worldwide concerned cancer. In spite of the advances in treatment, the 5-year survival rate has only increased subtly during the past two decades, which is largely due to the advanced stages of disease at diagnosis and the frequent development of relapse and second primary tumors. Therefore, the identification of underlying OSCC protein biomarker during cancer initiation and progression could aid the diagnosis and treatment of OSCC. In this review, recent researches on proteomics analysis of tissue, saliva, and serum for early detection and evaluation aggressiveness and occurrence of OSCC were summarized. The emphasis is placed on early detection by tissues, saliva, and serum of patients with histologically defined OSCC patients. Although lots of researches for searching OSCC protein biomarker have done, few common protein biomarkers have been detected. Low-redundant protein in tissues, saliva, and serum from OSCC may more accurately reflected the progression of OSCC, so novel approach for the depth research strategy and the sample choice for proteomics are of importance in OSCC biomarker discovery.     

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.