Clizia Chinello

Differential protein profiling of renal cell carcinoma urinary exosomes

Renal cell carcinoma (RCC) accounts for about 3% of all human malignancies and its incidence is increasing. There are no standard biomarkers currently used in the clinical management of patients with renal cell carcinoma. A promising strategy for new biomarker detection is comparative proteomics of urinary exosomes (UE), nanovesicles released by every epithelial cell facing the urinary space, enriched in renal proteins and excluding high-abundance plasmatic proteins, such as albumin. Aim of the work is to establish the protein profile of exosomes isolated from urines of RCC patient compared with control subjects. We enrolled 29 clear cell RCC patients and 23 control healthy subjects (CTRL), age and sex-matched, for urine collection and vesicle isolation by differential centrifugation. Such vesicles were morphologically and biochemically characterized and proved to share exosome properties. Proteomic analysis, performed on 9 urinary exosome (UE) pooled samples by gel based digestion followed by LC-MS/MS, led to the identification of 261 proteins from CTRL subject UE and 186 from RCC patient UE, and demonstrated that most of the identified proteins are membrane associated or cytoplasmic. Moreover, about a half of identified proteins are not shared between RCC and control UE. Starting from these observations, and from the literature, we selected a panel of 10 proteins, whose UE differential content was subjected to immunoblotting validation. Results show for the first time that RCC UE protein content is substantially and reproducibly different from control UE, and that these differences may provide clues for new RCC biomarker discovery.

Human urine biomarkers of renal cell carcinoma evaluated by ClinProt

Renal cell carcinoma (RCC) is one of the major causes of cancer death and is radio‐ and chemoresistant. Urine of 29 healthy subjects and 39 clear cell RCC patients were analyzed using the ClinProt technique to search for possible biomarkers for early RCC diagnosis. A cluster of three signals (marker A= at m/z 1827 ± 8 Da, marker B = 1914 ± 8 Da and marker C = 1968 ± 8 Da) was able to discriminate patients from controls. A receiver operating characteristic curve analysis showed values of area under the curve (AUC) higher than 0.9 for marker A and B, corresponding to a sensitivity of 85–90% and a specificity of 90%, while marker C gave a lower AUC (0.84) corresponding to sensitivity of 70% and specificity of 100%. The combination of three markers lead to an improvement in diagnostic efficacy, with specificity and sensitivity of 100% and 95%, respectively, in the training test and of 100% and of 85% in the test experiment. The efficacy of this cluster of signals to distinguish RCC patients grouped by tumor stage showed a sensibility of 100% for patients at the primary tumor 1 stage. One of the signals present in the cluster was identified as a fragment of Tamm‐Horsfall protein.

Detection of high molecular weight proteins by MALDI imaging mass spectrometry

MALDI imaging mass spectrometry (IMS) is a unique technology to explore the spatial distribution of biomolecules directly on tissues. It allows the in situ investigation of a large number of small proteins and peptides. Detection of high molecular weight proteins through MALDI IMS still represents an important challenge, as it would allow the direct investigation of the distribution of more proteins involved in biological processes, such as cytokines, enzymes, neuropeptide precursors and receptors. In this work we compare the traditional method performed with sinapinic acid with a comparable protocol using ferulic acid as the matrix. Data show a remarkable increase of signal acquisition in the mass range of 20k to 150k Th. Moreover, we report molecular images of biomolecules above 70k Th, demonstrating the possibility of expanding the application of this technology both in clinical investigations and basic science.

Alterations of the serum peptidome in renal cell carcinoma discriminating benign and malignant kidney tumors

Renal cell carcinoma (RCC) is typically asymptomatic and surgery usually increases patients life only for early stage tumors. However, some cystic and solid renal lesions cannot be confidently differentiated from clear-cell-RCC. Therefore possible markers for early detection and to distinguish malignant kidney tumors are needed. To this aim, we applied MALDI-TOF and LC-MS/MS analysis to RPC18 MB purified serum of ccRCC, non-ccRCC patients and controls. A cluster of five signals differentiate malignant tumors from benign renal masses and healthy subjects. Moreover, a combination of six ions showed the highest specificity and sensitivity to distinguish ccRCC from controls. Healthy subjects were also differentiated from non-ccRCC by three features. Peptide ratios obtained by MALDI-TOF were compared with those from label-free LC-ESI and no statistical difference was found (p>0.05). ESI-results were linked with MALDI profiles by both TOF/TOF sequencing and MALDI FT-ICR accurate mass measurements. About 200 unique endogenous peptides, originating from 32 proteins, were identified. Among them, SDPR and ZYX were found down-expressed, while SRGN and TMSL3 were up-expressed. In conclusion, our results suggest the possibility to discriminate malignant kidney tumors based on a cluster of serum peptides. Moreover, label-free approach may represent a valid method to verify results obtained by MALDI-TOF. This article is part of a Special Issue entitled: Integrated omics.

Primary Cell Cultures from Human Renal Cortex and Renal-Cell Carcinoma Evidence a Differential Expression of Two Spliced Isoforms of Annexin A3

Primary cell cultures from renal cell carcinoma (RCC) and normal renal cortex tissue of 60 patients have been established, with high efficiency (more than 70%) and reproducibility, and extensively characterized. These cultures composed of more than 90% of normal or tumor tubular cells have been instrumental for molecular characterization of Annexin A3 (AnxA3), never extensively studied before in RCC cells although AnxA3 has a prognostic relevance in some cancer and it has been suggested to be involved in the hypoxia-inducible factor-1 pathway. Western blot analysis of 20 matched cortex/RCC culture lysates showed two AnxA3 protein bands of 36 and 33 kDa, and two-dimensional Western blot evidenced several specific protein spots. In RCC cultures the 36-kDa isoform was significantly down-regulated and the 33-kDa isoform up-regulated. Furthermore, the inversion of the quantitative expression pattern of two AnxA3 isoforms in tumor cultures correlate with hypoxia-inducible factor-1alpha expression. The total AnxA3 protein is down-regulated in RCC cultures as confirmed also in tissues by tissue microarray. Two AnxA3 transcripts that differ for alternative splicing of exon III have been also detected. Real-time PCR quantification in 19 matched cortex/RCC cultures confirms the down-regulation of longer isoform in RCC cells. The characteristic expression pattern of AnxA3 in normal and tumor renal cells, documented in our primary cultures, may open new insight in RCC management.

Serum biomarkers of Renal Cell Carcinoma assessed using a protein profiling approach based on ClinProt technique

OBJECTIVES To investigate the possibility of using the ClinProt technique to find serum cancer related diagnostic markers that are able to better discriminate healthy subjects from patients affected by renal cell carcinoma (ccRCC). Renal cell carcinoma is the most common malignancy of the kidney. Biomarkers for early detection, prognosis, follow-up, and differential diagnosis of ccRCC from benign renal lesions are needed in daily clinical practice when imaging is not helpful. METHODS Serum of 29 healthy subjects and 33 ccRCC patients was analyzed by the ClinProt/MALDI-ToF technique. RESULTS A cluster of 3 peptides (A = m/z 1083 +/- 8 Da, B = m/z 1445 +/- 8 Da and C = m/z 6879 +/- 8 Da) was able to discriminate patients from control subjects. Cross-validation analysis using the whole casistic showed 88% and 96% of sensitivity and specificity, respectively. Moreover, the cluster showed 100% sensitivity for the identification of patients at pT2 (n = 5) and pT3 (n = 8) and 85% for pT1 patients (n = 20). The intensity of peaks A and C continuously decreased from pT1 to pT3, whereas peak B increased in pT1 and pT2. CONCLUSIONS These results may be useful to set up new diagnostic or prognostic tools.

Imaging mass spectrometry: a new tool for kidney disease investigations

Matrix-assisted laser desorption ionization (MALDI)-profiling and imaging mass spectrometry are promising technologies for measuring hundreds of different molecules directly on tissues. For instance, small molecules, drugs and their metabolites, endogenous lipids, carbohydrates and complex peptides/proteins can be measured at the same time without significant disruption of sample integrity. In this review, the potential of MALDI-profiling/imaging technologies in disease proteomics, drug action and studies of cellular processes in the context of kidney tissue is described. Spatial and sequence information obtained in tissue MALDI-profiling/imaging studies can be correlated with other mass spectrometry-based techniques, auxiliary imaging technologies and routine (immuno) histochemical staining.

Biomarkers discovery by peptide and protein profiling in biological fluids based on functionalized magnetic beads purification and mass spectrometry

Proteomics aims for the full identification and quantification of all expressed proteins in any organism. This is however an extremely tedious task since one gene often accounts for multiple proteins due to gene splicing and processing of proteins, such as the addition of post-translational modifications. Moreover, the concentration range of occurring proteins varies more than a factor of one million. For these reasons, protein profiling was considered a promising technique in the early days of proteomics. Ideally a protein profile can be observed in one single measurement. In various clinical studies profiling methods have been successful in the detection of proteome variations as a consequence of an altered homeostasis. Proteins that are differentially expressed as a consequence of a disease are very useful in medical science as they can be used as new biomarkers for the diagnosis, prognosis and as possible therapeutic targets. In order to find such proteins or biomarkers two different kinds of biological material have been used: tissue samples and body fluids. Tissues are obtained from biopsies, from stable cell lines or cell cultures, or from subcellular fractions. Despite their large usage tissues suffer from several disadvantages. Tissue samples are difficult to obtain and are comprised of several different type of cells. Standardization of the methods to obtain subcellular fraction that affects its preparation and purity is a challenge not yet solved. The difference between a cell culture and its corresponding wild type present in the body limits the translation of information derived from the first to the latter. On the contrary body fluids do not suffer from these limitations inherent to tissue samples. Fluids are very easily accessible with non- or very low-invasive methods at relatively low cost. They perfuse all the organs in the body carrying secreted protein from tissues. Therefore the protein profile of the biological fluids can reflect the status of the body. Among biological fluids serum, plasma and urine are the most analyzed samples but also cerebrospinal fluid (CSF), saliva, amniotic fluids have been used. Moreover classical methods to investigate the tissue proteome, aiming at biomarker discovery, are generally based on two-dimensional electrophoresis (2DE) and are not suitable for clinical chemistry lab requirements in which large sample cohorts have to be analyzed in a short time. This addresses another great potential of body fluids profiling: the analysis can be carried out high-throughput without sacrificing robustness and quality of the method. In fact 2DE is a laborious process that is difficult to automate. It still suffers from several technical limitations in terms of repeatability and reproducibility even though progress has been made using three different fluorescent labels that enables simultaneous migration of three samples on the same gel (e.g proteins extracted from control and disease, and the internal standard).Since the beginning of the 1990ties, when this new term (proteomics) was coined, a lot of progress has been made. Among them, several strategies to search these biomarkers in biological fluids have been developed in order to try to tackle some of the limitations of the current methods. Nowadays, mass spectrometry (MS) is the method of choice for the analysis of proteins, and as a consequence the field is now often referred to as MS-based proteomics. Direct analysis of the biological fluids with mass spectrometry is a challenging approach due to the sample complexity. To carry out a repeatable and robust mass spectrometric analysis of proteins in body fluids a suitable clean-up procedure is required in which salts and detergents are removed. The presence of salts can suppress the ionization in the mass spectrometer and chromatographic profiles may be influenced by from tailing due to co-elution of contaminants1. Therefore a pre-fractionation of the fluids is essential in order to increase the number of proteins that can be detected within a single MS-experiment, thus facilitating the discovery of new markers. Moreover, the fractionation of the biological fluids will also enrich low abundant proteins in fractions. These approaches lead to build the protein profile of the different biological fluids. Variations observed in patient profiles of body fluids compared to those of controls can be used to find the best pattern of signals that allows to discriminate two populations or to stratify the patients according to tumour stage or to the response to the therapy. One the major advantages of this strategy is that no pre-knowledge of the identity of signals selected for the cluster is needed to allow their use as biomarkers2. A specific agent to capture proteins enriches the sample and thus contributes to sensitivity enhancement. In general, protein separation techniques are based on different protein physical properties, such as size, isoelectric point, solubility and affinity. Materials known from different chromatographic platforms are coupled to the surface of a carrier in order to obtain peptides and proteins. One of the first approaches to pre-fractionate the body fluid proteome using an activated surface was the Surface-Enhanced Laser Desorption/Ionization (SELDI) technique. The SELDI technique for protein profiling is probably the most known and widely used approach in which biological fluids are applied directly to a target plate that is later introduced into a mass spectrometer. After removing unbound material to the modified surface of the SELDI chip, the molecular weight of the captured proteins on the target plate is determined using a time-of-flight (TOF) mass analyzer3. In this way the body fluid protein profile for the studied population is obtained. This technology is not free of criticism. In particular not very good reproducibility of the results due to drift, noise or the use of different lots of chips are reported. Moreover the direct identification of these markers cannot be carried out using the SELDI-TOF system. Their identity has to be determined with different analytical approaches. Promising alternatives to this technology are based on magnetic beads with a functionalized or activated surface or on miniaturized chromatographic systems that allow off-line fractionation of the proteome present in the fluids before MS analysis. The combination of magnetic bead purification and matrix-assisted laser desorption ionization (MALDI) TOF-MS has been shown a powerful alternative to the SELDI-platform: the active surface of magnetic beads is much larger, resulting in a higher binding capacity, and identification of captured peptides and protein is possible through the use of a more advanced TOF mass analyzer. Moreover, only a small part of the eluted peptide/proteins fractions are used for the protein profile and the remaining sample can be to use to identify markers with other MS-approaches (e.g. MALDI-TOF/TOF or LC-ESI-MS/MS) without the need of additional purification. This review is mainly focussed on the pre-fractionation based on magnetic beads and their applications.

Proteomic analysis in clear cell renal cell carcinoma: identification of differentially expressed protein by 2-D DIGE

Renal cell carcinoma (RCC), the most common neoplasm affecting the adult kidney, is characterised by heterogeneity of histological subtypes, drug resistance, and absence of molecular markers. Two-dimensional difference gel electrophoresis (2-D DIGE) technology in combination with mass spectrometry (MS) was applied to detect differentially expressed proteins in 20 pairs of RCC tissues and matched adjacent normal kidney cortex (ANK), in order to search for RCC markers. After gel analysis by DeCyder 6.5 and EDA software, differentially expressed protein spots were excised from Deep Purple stained preparative 2DE gel. A total of 100 proteins were identified by MS out of 2500 spots, 23 and 77 of these were, respectively, over- and down-expressed in RCC. The Principal Component Analysis applied to gels and protein spots exactly separated the two sample classes in two groups: RCC and ANK. Moreover, some spots, including ANXA2, PPIA, FABP7 and LEG1, resulted highly differential. The DIGE data were also confirmed by immunoblotting analysis for these proteins. In conclusion, we suggest that applying 2-D DIGE to RCC may provide the basis for a better molecular characterization and for the discovery of candidate biomarkers.

Different expression of fibrinopeptide A and related fragments in serum of type 1 diabetic patients with nephropathy.

Type 1 diabetes (insulin-dependent diabetes mellitus, IDDM) is an autoimmune disease affecting about 0.12% of the worlds population. Diabetic nephropathy (DN) is a major long-term complication of both types of diabetes and retains a high human, social and economic cost. Thus, the identification of markers for the early detection of DN represents a relevant target of diabetic research. The present work is a pilot study focused on proteomic analysis of serum of controls (n=9), IDDM patients (n=10) and DN patients (n=4) by the ClinProt profiling technology based on mass spectrometry. This approach allowed to identify a pattern of peptides able to differentiate the studied populations with sensitivity and specificity close to 100%. Variance of the results allowed to estimate the sample size needed to keep the expected False Discovery Rate low. Moreover, three peptides differentially expressed in the serum of patients as compared to controls were identified by LC-ESI MS/MS as the whole fibrinopeptide A peptide and two of its fragments, respectively. The two fragments were under-expressed in diabetic patients, while Fibrinopeptide A was over-expressed, suggesting that anomalous turnover of Fibrinopeptide A could be involved in the pathogenesis of DN.