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Objective: Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is a unique proteomic technology that explores the spatial distribution of biomolecules directly in situ, thus integrating molecular and morphological information. The possibility of correlating distribution maps of multiple analyses with cytological features makes it an ideal research tool for discovering new diagnostic markers. A previous study showed that MALDI-IMS could help discrimination between different types of thyroid lesions, especially papillary thyroid carcinoma (PTC); the present feasibility study on ex vivo fine needle aspiration (FNA) smears describes its potential in detecting new proteomic targets of other thyroid lesions (follicular lesions, medullary carcinoma). Methods: MALDI-IMS was conducted on ex vivo FNAs obtained from surgical specimens and corresponding in vivo samples. Differences between proteomic profiles of different thyroid lesions were compared. Results: Comparing the protein profiles of hyperplastic nodules obtained from three different patients with each other, and with a new PTC, showed a high degree of concordance, indicating good reproducibility of the IMS technology on cytological samples, suggesting its potential as a tool for biomarker discovery. Furthermore, comparison of the average proteomic profiles of hyperplastic nodules with a Hürthle cell adenoma revealed significant differences, underlying the capability of MALDI-IMS to distinguish between different thyroid lesions. Finally, the proteomic profile of medullary thyroid carcinoma was also characterized. Conclusions: Our results confirmed the possible role of MALDI-IMS in the search for diagnostic targets of PTC and follicular lesions, which could be applied in larger trials aimed at the identification of proteins, convertible to cost-effective diagnostic tools such as immunohistochemistry. These tests could be used to analyse in vivo cytological smears, improving the preoperat

Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF)—profiling and imaging mass spectrometry (MSI) 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. In the most advanced instruments, it is achieved without significant disruption of sample integrity. MSI is a unique approach for assessing the spatial distribution of molecules using graphical multidimensional maps of their constituent analytes, which may for instance be correlated with histopathological alterations in patient tissues. MALDI-TOFMSI technology has been implemented in hospitals of several countries, where it is routinely used for quick pathogen(s) identification, a task formerly accomplished by laborious and expensive DNA/RNA-based PCR (polymerase chain reaction) screening.In this chapter, we describe how MSI is performed, what is required from the researcher, the instrument vendors and finally what can be achieved with MSI. We restrict our descriptions only to MALDI- MSI although several other MS techniques of ionization can easily be linked to MSI.

Aims: The in-situ proteomics technology known as matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) is a powerful technique that combines traditional histology and proteomics. Methods and results: MALDI-IMS was applied to routine diagnostic kidney biopsies in a small group of cases of membranous glomerulonephritis and minimal change disease. Molecular changes were observed not only in the tissue areas with pathological alterations, but also in morphologically normal-looking tissue, highlighting the potential feasibility of using MALDI-IMS as a tool in nephropathology. Conclusions: This technology can be applied to any biopsy where a frozen section is obtained as part of the diagnostic process. Although we do not yet know the molecular identity of the differentially expressed proteins/peptides, they could represent powerful classifiers of nosological groups. © 2013 John Wiley & Sons Ltd.

Preterm delivery is one of the main causes of perinatal morbidity and mortality and it accounts for 75 % of perinatal mortality and more than half of the long-term morbidity. We applied a proteomic approach based on mass spectrometry (MS) for biomarkers discovery of preterm premature rupture of membranes (pPROM) by investigating amniotic fluid (AF) invasively and non-invasively collected

Matrix-assisted laser desorption/ionization (MALDI) and mass spectrometry (MS) imaging are advanced technologies capable of revealing the spatial distribution of different molecules--e.g., drugs and their metabolites, endogenous lipids and complex peptides/proteins--directly in tissue specimens at the same time. Information obtained regarding tissues by MALDI profiling/imaging analysis can be correlated with other MS-based techniques, auxiliary imaging technologies and routine immunohistochemical stainings. In this review we describe the MALDI profiling/imaging technologies, providing examples of their application in kidney research.

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.

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.

Renal cell carcinoma (RCC) is typically asymptomatic and surgery usually increases patient's 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.

Purpose:To investigate the use of ClinProt technique to identify cancer markers in plasma of patients suffering from squamous cell carcinoma of the penis (SCCP). Materials and Methods:Plasma of 36 healthy subjects and 25 patients with penile carcinoma who underwent surgical treatment between June 2010 and June 2011 was collected and analyzed by the ClinProt/MALDI/ToF technique. Then the peptides were identified from the C8 MB eluted fraction of patients' and control subjects' plasma by LIFT MS/MS. Results:A cluster of 2 peptides (A=m/z 1897.22 ± 9 Da and B=m/z 2021.99 ± 9 Da) was able to discriminate patients from control subjects. Cross validation analysis using the whole casuistic showed 62.5% and 86.76% sensitivity and specificity, respectively. The cluster also showed very high sensitivity (100%) and specificity (97%) for SCCP patients that died due to the disease. Furthermore, patients with lymph node involvement presented sensitivity and specificity of 80% and 97%, respectively. These two peptides were identified by the proteomic approach based on a MALDI-TOF/TOF as fragments of C3 (m/z 1896.17) and C4a/b (m/z 2021.26) complement proteins. Conclusions:The results showed that as the disease progresses, the fragments C3 and C4 A/B are less expressed in comparison with healthy subjects. These results may be useful as prognostic tools

The exposure of healthy subjects to high altitude represents a model to explore the pathophysiology of diseases related to tissue hypoxia and to evaluate pharmacological approaches potentially useful as a therapy for chronic diseases related to hypoxia. We explored the urinary peptidome to detect alterations induced by the exposure of subjects to different altitudes (sea level, high altitude = 3500 m, very high altitude = 5400 m) and to pharmacological treatment. Urine samples were collected from 47 subjects, randomly and blindly assigned to placebo (n = 24) or Telmisartan (n = 23). Samples were purified by the use of magnetic beads, then analysed by MALDI-TOF MS. Results showed that the urinary peptidome is not affected by the administration of Telmisartan, neither at the sea level nor at high and very high altitudes. In contrast, the urinary protein profiles are modified when subjects are exposed to high and very high altitudes, and we detected six peptides differentially expressed in hypobaric hypoxia at high or very high altitude compared to the sea level. Two of them were identified as fragments of the glycoprotein uromodulin and of the a1-antitrypsin. This is the first proteomic study showing that hypobaric hypoxia conditions affect the urinary peptidome.

"Signal alignments play critical roles in many clinical setting. This is the case of mass spectrometry (MS) data, an important component of many types of proteomic analysis. A central problem occurs when one needs to integrate (MS) data produced by different sources, e.g., different equipment and/or laboratories. In these cases, some form of "data integration or "data fusion may be necessary in order to discard some source-specific aspects and improve the ability to perform a classification task such as inferring the "disease classes of patients. The need for new high-performance data alignments methods is therefore particularly important in these contexts. In this paper, we propose an approach based both on an information theory perspective, generally used in a feature construction problem, and the application of a mathematical programming task (i.e., the weighted bipartite matching problem). We present the results of a competitive analysis of our method against other approaches. The analysis was conducted on data from plasma/ethylenediaminetetraacetic acid of "control and Alzheimer patients collected from three different hospitals. The results point to a significant performance advantage of our method with respect to the competing ones tested. © 2012 IEEE.

The PhD thesis is focused on the evaluation of different Mass Spectrometry approaches for the study of the proteome of biological fluids and tissues. In detail, the ClinProt technology has been applied to amniotic fluids and urines respectively, to evaluate potential biomarkers for the preterm premature rupture of the membranes (pPROM) and to invetsigate molecular mechanisms of kidney adaptation to hypobaric hypoxia conditions at high and very high altitude. MALDI Imaging Mass Spectrometry (IMS) has been applied for the study of tissues. In detail, this part of the work evaluated the use of the detergents to enhance sensitivity and number of peaks detected for protein IMS analysis.

Iron is tightly connected to oxygen homeostasis and erythropoiesis. Our aim was to better understand how hypoxia regulates iron acquisition for erythropoiesis in humans, a topic relevant to common hypoxia-related disorders. Fortyseven healthy volunteers participated in the HIGHCARE project. Blood samples were collected at sea level and after acute and chronic exposure to high altitude (3400-5400 m above sea level). We investigated the modifications in hematocrit, serum iron indices, erythropoietin, markers of erythropoietic activity, interleukin-6, and serum hepcidin. Hepcidin decreased within 40 hours after acute hypoxia exposure (P < .05) at 3400 m, reaching the lowest level at 5400 m (80% reduction). Erythropoietin significantly increased (P < .001) within 16 hours after hypoxia exposure followed by a marked erythropoietic response supported by the increased iron supply. Growth differentiation factor-15 progressively increased during the study period. Serum ferritin showed a very rapid decrease, suggesting the existence of hypoxia-dependent mechanism(s) regulating storage iron mobilization. The strong correlation between serum ferritin and hepcidin at each point during the study indicates that iron itself or the kinetics of iron use in response to hypoxia may signal hepcidin down-regulation. The combined and significant changes in other variables probably contribute to the suppression of hepcidin in this setting. © 2011 by The American Society of Hematology.

Matrix-Assisted Laser Desorption/Ionization (MALDI) Imaging Mass Spectrometry (IMS) is a molecular technology that allows simultaneous investigation of the content and spatial distribution of molecules within tissue. In this work, we examine different classes of detergents, the anionic sodium dodecyl sulfate (SDS), the nonionic detergents Triton X-100, Tween 20 and Tween 80, and the zwitterionic 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) for use in MALDI IMS of analytes above m/z 4000. These detergents were found to be compatible with MALDI MS and did not cause signal suppression relative to non-detergent applications and did not produce interfering background signals. In general, these detergents enhanced signal acquisition within the mass range m/z 4-40 000. Adding detergents into the matrix was comparable with the separate application of detergent and matrix. Evaluation of spectra collected from organ-specific regions of a whole mouse pup section showed that different detergents perform optimally with different organs, indicating that detergent selection should be optimized on the specific tissue for maximum gain. These data show the utility of detergents towards enhancement of protein signals for on-tissue MALDI IMS analysis.

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.

Type 1 diabetes (insulin-dependent diabetes mellitus, IDDM) is an autoimmune disease affecting about 0.12% of the world's 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. © 2009 Elsevier B.V. All rights reserved.

Clinical data alignment plays a critical role in identifying important features for significant experiments. A central problem is data fusion i.e., how to correctly integrate data provided by different labs. This integration is done in order to increase ability of inferring target classes of controls and patients. Our paper proposes an approach based both on a information theoretic perspective, generally used in a feature construction problem [3] and on the approximated solution for a mathematical programming task (i.e. the weighted bipartite matching problem [6]). Numerical evaluations with two competitive approaches show the improved performance of the proposed method. For this evaluation we used data sets from plasma / ethylenediaminetetraacetic acid (EDTA) of controls and Alzheimer patients collected in three different hospitals.

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 ofthe signals present in the cluster was identified as a fragment of Tamm-Horsfall protein. © 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.