Discovering cancer biomarkers from clinical samples by protein microarrays

Cancer biomarkers are of potential use in early cancer diagnosis, anticancer therapy development, and monitoring the responses to treatments. Protein-based cancer biomarkers are major forms in use, as they are much easier to be monitored in body fluids or tissues. For cancer biomarker discovery, high-throughput techniques such as protein microarrays hold great promises, because they are capable of global unbiased monitoring but with a miniaturized format. In doing so, novel and cancer type specific biomarkers can be systematically discovered at an affordable cost. In this review, we give a relatively complete picture on protein microarrays applied to clinical samples for cancer biomarker discovery, and conclude this review with the future perspectives.     

Relationship between exo‐electron currents from MgO thin film and statistical delay time in ACPDPs

Abstract— The exo‐electron currents from a ACPDP test panel with or without MgO crystals sprayed on MgO film were measured directly after eliminating of the wall‐voltage effect. An inverse relationship was established between the statistical delay time and exo‐electron currentfrom the MgO cathode film. The spraying of MgO crystals on MgO thin film was observed to reduce the statistical delay time dramatically even for the same exo‐electron currents measured. The shift of the inverse curve may be attributed to an increased discharge success probability by the MgO crystals sprayed.     

Differential expression of ceruloplasmin isoforms in the cerebrospinal fluid of amyotrophic lateral sclerosis patients

Amyotrophic lateral sclerosis (ALS) a fatal degenerative disease that selectively affects motor neurons, likely results from a complex interplay among oxidative injury, excitotoxic stimulation, protein aggregation and genetic factors. Ceruloplasmin (Cp) protein is a ferroxidase that oxidizes toxic ferrous iron to its nontoxic ferric form, protecting the central nervous system (CNS) from iron deposition. Cp is thus considered as one of the main systems dedicated to the protection of the CNS from oxidative stress damage. We investigated Cp protein behaviour in the cerebrospinal fluid (CSF) of ALS patients of recent onset. An increased expression of Cp was observed in ALS (n = 16) compared to two control groups (healthy subjects, n = 11 and peripheral neuropathy patients, n = 10). 2‐DE analysis revealed a differential expression of Cp isoforms in ALS patients compared to controls. ALS samples showed an increase in the relative abundance of more basic Cp forms, corresponding to the nonsialylated proteins. Despite the increase in protein expression, ferroxidase activity evaluated in the CSF of ALS patients was comparable to that of the controls, indicating a Cp functional impairment. Ceruloplasmin isoforms profile may be proposed as disease feature that could provide insight into the molecular mechanisms of ALS pathogenesis.     

Differential protein expression between normal, early-stage, and late-stage myxomatous mitral valves from dogs

Valvular heart disease accounts for over 20 000 deaths and 90 000 hospitalizations yearly in the United States. Myxomatous valve disease (MVD) is the most common disease of the mitral valve in humans and dogs. MVD is pathologically identical in these species and its pathogenesis is poorly understood. The objectives of this study were to (i) develop proteomic methodology suitable for analysis of extracellular matrix‐rich heart valve tissues and (ii) survey over‐ and under‐expressed proteins that could provide mechanistic clues into the pathogenesis of MVD. Normal, early‐stage, and late‐stage myxomatous mitral valves from dogs were studied. A shotgun proteomic analysis was used to quantify differential protein expression. Proteins were classified by function and clustered according to differential expression patterns. More than 300 proteins, with 117 of those being differentially expressed, were identified. Hierarchical sample clustering of differential protein profiles showed that early‐ and late‐stage valves were closely related. This finding suggests that proteome changes occur in early degeneration stages and these persist in late stages, characterizing a diseased proteome that is distinct from normal. Shotgun proteome analysis of matrix‐rich canine heart valves is feasible, and should be applicable to human heart valves. This study provides a basis for future investigations into the pathogenesis of MVD.     

Differential expression of AQP1 in microdomain-enriched membranes of renal cell carcinoma

Human aquaporin-1 (AQP1) is the most studied member of the aquaporin family, acting as molecular water channel. It is also considered a differentiation marker for proximal renal tubular cells, from which clear cells renal cell carcinoma (RCC) originates, playing an important role in urine formation. We therefore studied AQP1 expression at the proteomic level in RCC and normal tissues, mainly focusing on microdomain-enriched membranes in which AQP1 is highly concentrated. Subcellular fractions were prepared through differential centrifugation, and microdomain-enriched fractions were purified from a plasma membrane-enriched fraction by 1% Triton X-100 treatment followed by ultracentrifugation in sucrose gradient. After SDS-PAGE and Western blot analyses with antibodies against AQP1, lower expression levels of AQP1 isoforms were observed in each subcellular fraction of RCC compared to fractions from normal kidney tissues. The presence of AQP1 in the immunoreactive bands was verified by MALDI-TOF-MS and LC-ESI-MS/MS analysis. Glycosylation of AQP1 was also investigated using N-glycosidase F, confirming the presence of a N-glycosylated isoform of AQP1 in the 35-45-kDa region. These results highlight an under-expression of AQP1 protein and its glycosylated isoforms in homogenate and subcellular fraction obtained from RCC tissue compared to adjacent normal cortex.     

Identification of distinctive protein expression patterns in colorectal adenoma

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

Aberrant protein expression in plasma and kidney tissue during experimental obstructive nephropathy

Kidney failure is a major health problem worldwide. Patients with end-stage renal disease require intensive medical support by dialysis or kidney transplantation. Current methods for diagnosis of kidney disease are either invasive or insensitive, and renal function may decline by as much as 50% before it can be detected using current techniques. The goal of this study was, therefore, to identify biomarkers of kidney disease (associated with renal fibrosis) that can be used for the development of a non-invasive clinical test for early disease detection. We utilized two protein-profiling technologies (SELDI-TOF MS and 2-D) to screen the plasma and kidney proteome for aberrantly expressed proteins in an experimental mouse model of unilateral uretric obstruction, which mimics the pathology of human renal disease. Several differentially regulated proteins were detected at the plasma level of day-3-obstructed animals, which included serum amyloid A1, fibrinogen α, haptoglobin precursor protein, haptoglobin and major urinary proteins 11 and 8. Differentially expressed proteins detected at the tissue level included ras-like activator protein 2, haptoglobin precursor protein, malate dehydrogenase, α enolase and murine urinary protein (all p<0.05 versus controls). Immunohistochemistry was used to confirm the up-regulation of fibrinogen. Interestingly, these proteins are largely separated into four major classes: (i) acute-phase reactants (ii) cell-signaling molecules (iii) molecules involved in cell growth and metabolism and (iv) urinary proteins. These results provide new insights into the pathology of obstructive nephropathy and may facilitate the development of specific assay(s) to detect and monitor renal fibrosis.     

Abnormal cytoskeletal protein expression in cultured skin fibroblasts from type 1 diabetes mellitus patients with nephropathy: A proteomic approach

Diabetic nephropathy (DN) develops in about 40% of insulin-dependent type 1 diabetes mellitus (T1DM) patients, and is associated not only with diabetes duration and metabolic control, but also with a genetic predisposition. Constitutive alterations of cytoskeletal proteins may play a role in the development of DN. We investigated the expression of these proteins in cultured skin fibroblasts, obtained from long-term T1DM patients with and without DN but comparable metabolic control, and from matched healthy subjects, by means of 2-DE electrophoresis and MS-MALDI analyses. In T1DM with DN, compared to the other two groups, quantitative analyses revealed an altered expression of 17 spots (p<0.05-p<0.01), corresponding to 12 unique proteins. In T1DM with DN, beta-actin and three isoforms of tubulin beta-2 chain, tropomodulin-3, and LASP-1 were decreased, whereas two tubulin beta-4 chain isoforms, one alpha actinin-4 isoform, membrane-organizing extension spike protein (MOESIN), FLJ00279 (corresponding to a fragment of myosin heavy chain, non-muscle type A), vinculin, a tropomyosin isoform, and the macrophage capping protein were increased. A shift in caldesmon isoforms was also detected. These results demonstrate an association between DN and the constitutive expression of cytoskeleton proteins in cultured skin fibroblasts from T1DM with DN, which may retain pathophysiologycal implications.     

Thymosin β4 is differentially expressed in the cerebrospinal fluid of Creutzfeldt‐Jakob disease patients: a MALDI‐TOF MS protein profiling study

MALDI‐TOF protein profiling analysis permits the detection of peptides and small proteins in complex protein mixtures with great accuracy. We applied this analysis to cerebrospinal fluid (CSF) from 15 patients affected by Creutzfeldt‐Jakob disease (CJD). We compared the levels of the normalized ion signals of 11 sporadic and 4 genetic CJD forms with those from ten healthy control subjects and eight non‐CJD relapsing‐remitting multiple sclerosis patients. In so doing, we detected 61 differentially expressed ion signals in CJD samples compared to controls. Among the 61 signals, 3 signals had significantly increased levels with high statistical significance (p <0.0001) and were located at 3238.3 m/z, 4963.7 m/z, and 8565.3 m/z. We characterized the 5.0 and 8.6 kDa proteins as thymosin β4 N‐acetylated and free ubiquitin, respectively, while the 3.2‐kDa peptide remained uncharacterized. Although elevated ubiquitin levels have previously been described in CJD, we have demonstrated for the first time the involvement of thymosin β4 in a neurodegenerative disease. To support the validity of thymosin β4 levels obtained by MALDI‐TOF analysis, an independent enzyme immunoassay analysis was performed. Moreover, a validation cohort consisting of CSF from three CJD patients, five healthy subjects, and six non‐CJD relapsing‐remitting multiple sclerosis patients was analyzed in a similar way, yielding superimposable results. We propose that thymosin β4 is a potential new candidate marker for the ante mortem diagnosis of CJD disease.     

Protein content in aqueous humor from patients with pseudoexfoliation (PEX) investigated by capillary LC MALDI‐TOF/TOF MS

Analysis of proteins in human body fluids is challenging since the composition of the sample often is rather complex. Here we present a method for analysis of proteins in aqueous humor from two groups of cataract patients, with and without pseudoexfoliation (PEX). Aqueous humor is an extracellular fluid contained in the anterior chamber of the eye between the cornea and iris. The limited volume of sample requires sophisticated analysis techniques. Our method is based on a total tryptic digestion of the sample followed by capillary LC‐MALDI MS and MS/MS analysis of the peptides. The method is rapid, efficient and suitable as a complement or alternative to more commonly used methods based on gel electrophoretic experiments. With this method we found and unambiguously identified 30 nonredundant proteins. Proteins found include general transport proteins such as albumin and apolipoprotein A1 but also specific proteins involved in immune response, such as complement factors. Cystatin C, clusterin, and crystallins were also found. Although the number of proteins was roughly the same in both groups there was a significant difference in their identities. These findings may give some new insights into the pathophysiology of the PEX syndrome.