New orphan disease therapies from the proteome of industrial plasma processing waste- a treatment for aceruloplasminemia.

Plasma-derived therapeutic proteins are produced through an industrial fractionation process where proteins are purified from individual intermediates, some of which remain unused and are discarded. Relatively few plasma-derived proteins are exploited clinically, with most of available plasma being directed towards the manufacture of immunoglobulin and albumin. Although the plasma proteome provides opportunities to develop novel protein replacement therapies, particularly for rare diseases, the high cost of plasma together with small patient populations impact negatively on the development of plasma-derived orphan drugs. Enabling therapeutics development from unused plasma fractionation intermediates would therefore constitute a substantial innovation. To this objective, we characterized the proteome of unused plasma fractionation intermediates and prioritized proteins for their potential as new candidate therapies for human disease. We selected ceruloplasmin, a plasma ferroxidase, as a potential therapy for aceruloplasminemia, an adult-onset ultra-rare neurological disease caused by iron accumulation as a result of ceruloplasmin mutations. Intraperitoneally administered ceruloplasmin, purified from an unused plasma fractionation intermediate, was able to prevent neurological, hepatic and hematological phenotypes in ceruloplasmin-deficient mice. These data demonstrate the feasibility of transforming industrial waste plasma fraction into a raw material for manufacturing of new candidate proteins for replacement therapies, optimizing plasma use and reducing waste generation.

Lipid dysmetabolism in ceruloplasmin-deficient mice revealed both in vivo and ex vivo by MRI, MRS and NMR analyses.

Ceruloplasmin (Cp) is a ferroxidase that plays a role in cellular iron homeostasis and is mainly expressed in the liver and secreted into the blood. Cp is also produced by adipose tissue, which releases it as an adipokine. Although a dysfunctional interaction of iron with the metabolism of lipids has been associated with several metabolic diseases, the role of Cp in adipose tissue metabolism and in the interplay between hepatocytes and adipocytes has been poorly investigated. We previously found that Cp-deficient (CpKO) mice become overweight and demonstrate adipose tissue accumulation together with liver steatosis during aging, suggestive of lipid dysmetabolism. In the present study, we investigated the lipid alterations which occur during aging in adipose tissue and liver of CpKO and wild-type mice both in vivo and ex vivo. During aging of CpKO mice, we observed adipose tissue accumulation and liver lipid deposition, both of which are associated with macrophage infiltration. Liver lipid deposition was characterized by accumulation of triglycerides, fatty acids and ω-3 fatty acids, as well as by a switch from unsaturated to saturated fatty acids, which is characteristic of lipid storage. Liver steatosis was preceded by iron deposition and macrophage infiltration, and this was observed to be already occurring in younger CpKO mice. The accumulation of ω-3 fatty acids, which can only be acquired through diet, was associated with body weight increase in CpKO mice despite food intake being equal to that of wild-type mice, thus underlining the alterations in lipid metabolism/catabolism in Cp-deficient animals.

Ceruloplasmin-Deficient Mice Show Dysregulation of Lipid Metabolism in Liver and Adipose Tissue Reduced by a Protein Replacement.

Ceruloplasmin is a ferroxidase that plays a role in iron homeostasis; its deficiency fosters inter alia iron accumulation in the liver, which expresses the soluble form of the protein secreted into the bloodstream. Ceruloplasmin is also secreted by the adipose tissue, but its role in adipocytes has been poorly investigated. We hypothesized that ceruloplasmin might have a role in iron/lipid interplay. We investigated iron/lipid dysmetabolism in the liver and adipose tissue of the ceruloplasmin-deficient mouse (CpKO) model of aceruloplasminemia and evaluated the effectiveness of ceruloplasmin replacement. We found that CpKO mice were overweight, showing adipose tissue accumulation, liver iron deposition and steatosis. In the adipose tissue of CpKO mice, iron homeostasis was not altered. Conversely, the levels of adiponectin and leptin adipokines behaved opposite to the wild-type. Increased macrophage infiltration was observed in adipose tissue and liver of CpKO mice, indicating tissue inflammation. The treatment of CpKO mice with ceruloplasmin limited liver iron accumulation and steatosis without normalizing the expression of iron homeostasis-related proteins. In the CpKO mice, the protein replacement limited macrophage infiltration in both adipose and hepatic tissues reduced the level of serum triglycerides, and partially recovered adipokines levels in the adipose tissue. These results underline the link between iron and lipid dysmetabolism in ceruloplasmin-deficient mice, suggesting that ceruloplasmin in adipose tissue has an anti-inflammatory role rather than a role in iron homeostasis. Furthermore, these data also indicate that ceruloplasmin replacement therapy may be effective at a systemic level.

Serological proteome analysis identifies crustacean myosin heavy chain type 1 protein and house dust mite Der p 14 as cross-reacting allergens.

BACKGROUND: Allergies to house dust mite (HDM) and to crustaceans are clinically and pathogenically linked. Several homologous allergenic proteins have been identified, among which tropomyosin is the prototype, expressing epitopes endowed with variable levels of immunoglobulin E (IgE) cross-reactivity. Component-resolved diagnosis (CRD) does not allow a thorough characterization of all relevant IgE reactivities to these allergen sources. OBJECTIVES: We studied 1 patient allergic to shrimp with positive skin prick test to HDM and negative scores for IgE to HDM allergen components routinely used in CRD (group 1 and 2 allergens, Der p 23 and tropomyosin). MATERIAL AND METHODS: In order to identify the allergen(s) involved in IgE reactivity, we used serological proteome analysis (SERPA), which utilizes two-dimensional gel electrophoresis (2DE), immunoblotting and mass spectrometry (MS). The identified allergenic proteins were tested with sera from 20 crustacean-allergic patients and 19 grass-allergic patients serving as controls. RESULTS: Der p 14 and myosin heavy chain type 1 (MHC1) were identified as the components recognized by patient's IgE in the proteome of Dermatophagoides pteronyssinus and Penaeus monodon, respectively. The MHC1 protein shows about 30% sequence identity with Der p 14 in specific domains, and cross-reactivity against epitopes shared by the 2 proteins was demonstrated by reduced reactivity to shrimp extract following pre-incubation with Der p 14. Serum IgE from 5 out of 20 patients allergic to crustaceans reacted with MHC1, compared to none among 19 controls (p < 0.05). CONCLUSION: We identified MHC1 as a relevant allergic component in the proteome of Penaeus monodon, the prototypic allergen source used in diagnosis of allergy to crustaceans. Our data demonstrate MHC1 cross-reactivity between MHC1 and Der p 14 from Dermatophagoides pteronyssinus.

Oxidized/deamidated-ceruloplasmin dysregulates choroid plexus epithelial cells functionality and barrier properties via RGD-recognizing integrin binding.

Choroid plexus epithelial cells (CPEpiCs) determine the composition of cerebrospinal fluid (CSF) and constitute the blood-CSF barrier (BCSFB), functions that are altered in neurodegenerative diseases. In Parkinson's disease (PD) the pathological environment oxidizes and deamidates the ceruloplasmin, a CSF-resident ferroxidase, which undergoes a gain of RGD-recognizing integrin binding property, that may result in signal transduction. We investigated the effects that oxidized/deamidated ceruloplasmin (Cp-ox/de) may exert on CPEpiCs functions. Through RGD-recognizing integrins binding, Cp-ox/de mediates CPEpiCs adhesion and intracellular signaling, resulting in cell proliferation inhibition and alteration of the secretome profile in terms of proteins related to cell-extracellular matrix interaction. Oxidative conditions, comparable to those found in the CSF of PD patients, induced CPEpiCs barrier leakage, allowing Cp-ox/de to cross it, transducing integrins-mediated signal that further worsens BCSFB integrity. This mechanism might contribute to PD pathological processes altering CSF composition and aggravating the already compromised BCSFB function.

ADAM10 Site-Dependent Biology: Keeping Control of a Pervasive Protease.

Enzymes, once considered static molecular machines acting in defined spatial patterns and sites of action, move to different intra- and extracellular locations, changing their function. This topological regulation revealed a close cross-talk between proteases and signaling events involving post-translational modifications, membrane tyrosine kinase receptors and G-protein coupled receptors, motor proteins shuttling cargos in intracellular vesicles, and small-molecule messengers. Here, we highlight recent advances in our knowledge of regulation and function of A Disintegrin And Metalloproteinase (ADAM) endopeptidases at specific subcellular sites, or in multimolecular complexes, with a special focus on ADAM10, and tumor necrosis factor-α convertase (TACE/ADAM17), since these two enzymes belong to the same family, share selected substrates and bioactivity. We will discuss some examples of ADAM10 activity modulated by changing partners and subcellular compartmentalization, with the underlying hypothesis that restraining protease activity by spatial segregation is a complex and powerful regulatory tool.

Ceruloplasmin Deamidation in Neurodegeneration: From Loss to Gain of Function.

Neurodegenerative disorders can induce modifications of several proteins; one of which is ceruloplasmin (Cp), a ferroxidase enzyme found modified in the cerebrospinal fluid (CSF) of neurodegenerative diseases patients. Cp modifications are caused by the oxidation induced by the pathological environment and are usually associated with activity loss. Together with oxidation, deamidation of Cp was found in the CSF from Alzheimer's and Parkinson's disease patients. Protein deamidation is a process characterized by asparagine residues conversion in either aspartate or isoaspartate, depending on protein sequence/structure and cellular environment. Cp deamidation occurs at two Asparagine-Glycine-Arginine (NGR)-motifs which, once deamidated to isoAspartate-Glycine-Arginine (isoDGR), bind integrins, a family of receptors mediating cell adhesion. Therefore, on the one hand, Cp modifications lead to loss of enzymatic activity, while on the other hand, these alterations confer gain of function to Cp. In fact, deamidated Cp binds to integrins and triggers intracellular signaling on choroid plexus epithelial cells, changing cell functioning. Working in concert with the oxidative environment, Cp deamidation could reach different target cells in the brain, altering their physiology and causing detrimental effects, which might contribute to the pathological mechanism.

Ceruloplasmin oxidized and deamidated by Parkinson's disease cerebrospinal fluid induces epithelial cells proliferation arrest and apoptosis.

In Parkinson's disease, the ferroxidase ceruloplasmin (Cp) is oxidized and deamidated by the pathological cerebrospinal fluid (CSF) environment. These modifications promote the gain of integrin binding properties, fostered by the deamidation of two NGR-motifs present in the Cp sequence that convert into the isoDGR-motif. Through isoDGR/integrin binding, the oxidized/deamidated-Cp (Cp-ox/de) mediates cell adhesion and transduces an intracellular signal in epithelial cells that seems to be addressed to regulate cell cycle, proliferation and cytoskeletal re-arrangement. However, the effect fostered on cells by integrins engagement via Cp-ox/de is not known. We found that in HaCaT epithelial cells, the incubation with Cp-ox/de resulted in proliferation inhibition mediated by isoDGR, cell cycle arrest and apoptosis induction. Similar proliferation inhibition was induced by treatment with purified Cp previously incubated in the CSF from Parkinson's disease patients, but not by Cp incubated in the CSF from healthy subjects. In human primary choroid plexus epithelial cells, a possible in vivo target of Cp-ox/de generated in pathological CSFs, we found that Cp-ox/de mediated cell adhesion via isoDGR/integrins binding and transduced an intracellular signal, which resulted in cell proliferation inhibition. Thus, the generation of Cp-ox/de in pathological CSFs and the consequent apoptosis induction of epithelial cells facing the liquor, might represent a novel mechanism that contributes to neurodegeneration.

Tuberomics: a molecular profiling for the adaption of edible fungi (Tuber magnatum Pico) to different natural environments.

BACKGROUND: Truffles are symbiotic fungi that develop underground in association with plant roots, forming ectomycorrhizae. They are primarily known for the organoleptic qualities of their hypogeous fruiting bodies. Primarily, Tuber magnatum Pico is a greatly appreciated truffle species mainly distributed in Italy and Balkans. Its price and features are mostly depending on its geographical origin. However, the genetic variation within T. magnatum has been only partially investigated as well as its adaptation to several environments. RESULTS: Here, we applied an integrated omic strategy to T. magnatum fruiting bodies collected during several seasons from three different areas located in the North, Center and South of Italy, with the aim to distinguish them according to molecular and biochemical traits and to verify the impact of several environments on these properties. With the proteomic approach based on two-dimensional electrophoresis (2-DE) followed by mass spectrometry, we were able to identify proteins specifically linked to the sample origin. We further associated the proteomic results to an RNA-seq profiling, which confirmed the possibility to differentiate samples according to their source and provided a basis for the detailed analysis of genes involved in sulfur metabolism. Finally, geographical specificities were associated with the set of volatile compounds produced by the fruiting bodies, as quantitatively and qualitatively determined through proton transfer reaction-mass spectrometry (PTR-MS) and gas-chromatography-mass spectrometry (GC-MS). In particular, a partial least squares-discriminant analysis (PLS-DA) model built from the latter data was able to return high confidence predictions of sample source. CONCLUSIONS: Results provide a characterization of white fruiting bodies by a wide range of different molecules, suggesting the role for specific compounds in the responses and adaptation to distinct environments.

Aceruloplasminemia: Waiting for an Efficient Therapy.

Aceruloplasminemia is an ultra-rare hereditary disorder caused by defective production of ceruloplasmin. Its phenotype is characterized by iron-restricted erythropoiesis and tissue iron overload, diabetes, and progressive retinal and neurological degeneration. Ceruloplasmin is a ferroxidase that plays a critical role in iron homeostasis through the oxidation and mobilization of iron from stores and subsequent incorporation of ferric iron into transferrin (Tf), which becomes available for cellular uptake via the Tf receptor. In addition, ceruloplasmin has antioxidant properties preventing the production of deleterious reactive oxygen species via the Fenton reaction. Some recent findings suggest that aceruloplasminemia phenotypes can be more heterogeneous than previously believed, varying within a wide range. Within this large heterogeneity, microcytosis with or without anemia, low serum iron and high serum ferritin, and diabetes are the early hallmarks of the disease, while neurological manifestations appear 10-20 years later. The usual therapeutic approach is based on iron chelators that are efficacious in reducing systemic iron overload. However, they have demonstrated poor efficacy in counteracting the progression of neurologic manifestations, and also often aggravate anemia, thereby requiring drug discontinuation. Open questions remain regarding the mechanisms leading to neurological manifestation and development of diabetes, and iron chelation therapy (ICT) efficacy. Recent studies in animal models of aceruloplasminemia support the possibility of new therapeutic approaches by parenteral ceruloplasmin administration. In this review we describe the state of the art of aceruloplasminemia with particular attention on the pathogenic mechanisms of the disease and therapeutic approaches, both current and perspective.

Phenotypic heterogeneity in seven Italian cases of aceruloplasminemia.

INTRODUCTION: Aceruloplasminemia is an ultra-rare hereditary disorder characterized by iron-restricted microcytic anemia and tissue iron overload associated with diabetes, retinal and progressive neurological degeneration. We describe genotypes and phenotypes at diagnosis, and disease evolution of seven Italian patients. METHODS: Anagraphical, biochemical, genetic, clinical and instrumental data were collected at diagnosis and during a long-term follow-up. Mutations, ferroxidase activity and Western Blot analysis of ceruloplasmin were performed according to standard protocols. RESULTS: Three mutations were already described (p.Phe217Ser, deletions of exon 11 and 12), p.Ile991Thr is a very rare variant, p.Cys338Ser and IVS6+1G > A were novel mutations. In silico analyses suggested they were highly likely or likely to be damaging. At diagnosis, 100% had microcytosis, 86% had mild-moderate anemia, low serum iron and high serum ferritin. Four (57%) had type 1 diabetes or glucose intolerance, 3/7 had neurological manifestations, and only one had early diabetic retinopathy. All but one underwent iron chelation therapy requiring temporary discontinuation because of anemia worsening. At the end of follow-up, three patients aggravated and 2 developed neurological symptoms; only two patients were free of neurological manifestations and showed mild or absent brain iron. CONCLUSION: Aceruloplasminemia phenotypes ranged from classical characterized by progressive neurologic derangement to milder in which signs of systemic iron overload prevailed over brain iron accumulation. Within this large heterogeneity, microcytosis with or without anemia, low serum iron and high serum ferritin were the early hallmarks of the disease. Therapeutic approaches other than iron chelation should be explored to reduce morbidity and improve life expectancy.

Ceruloplasmin replacement therapy ameliorates neurological symptoms in a preclinical model of aceruloplasminemia.

Aceruloplasminemia is a monogenic disease caused by mutations in the ceruloplasmin gene that result in loss of protein ferroxidase activity. Ceruloplasmin plays a role in iron homeostasis, and its activity impairment leads to iron accumulation in liver, pancreas, and brain. Iron deposition promotes diabetes, retinal degeneration, and progressive neurodegeneration. Current therapies mainly based on iron chelation, partially control systemic iron deposition but are ineffective on neurodegeneration. We investigated the potential of ceruloplasmin replacement therapy in reducing the neurological pathology in the ceruloplasmin-knockout (CpKO) mouse model of aceruloplasminemia. CpKO mice were intraperitoneal administered for 2 months with human ceruloplasmin that was able to enter the brain inducing replacement of the protein levels and rescue of ferroxidase activity. Ceruloplasmin-treated mice showed amelioration of motor incoordination that was associated with diminished loss of Purkinje neurons and reduced brain iron deposition, in particular in the choroid plexus. Computational analysis showed that ceruloplasmin-treated CpKO mice share a similar pattern with wild-type animals, highlighting the efficacy of the therapy. These data suggest that enzyme replacement therapy may be a promising strategy for the treatment of aceruloplasminemia.

Effects of different nitrogen fertilizers on two wheat cultivars: An integrated approach.

Investigation of cultivated plant physiology grown under low energy input plays an important role to indicate their fitness to the new environmental conditions. The durum-wheat cultivars Creso and Dylan were tested to evaluate the growth, production, and proteomic and transcriptomic profiles of the crop under different synthetic and organic nitrogen fertilization regimes. In this work, a two-dimensional gel electrophoresis (2-DE) approach combined with liquid chromatography-mass spectrometry (LC-MS) was used to investigate the protein changes induced by the use of different nitrogen sources (hydrolysate of proteins 1 and 2, rhizovit, synthesis, leather) on wheat plants. Proteomic studies were integrated with qPCR analysis of genes related to glutamine synthetase/glutamine-2-oxoglutarate aminotransferase (GS-GOGAT) and tricarboxylic acid (TCA) metabolic pathways because most relevant for nitrogen-dependent plants growth. The proteomic analysis lead to the isolation of 23 spots that were able to distinguish the analyzed samples. These spots yielded the identification of 60 proteins involved in photosynthesis, glycolysis, and nitrogen metabolism. As an example, the quinone oxidoreductase-like protein and probable glutathione S-transferase GSTU proteins were identified in two spots that represents the most statistically significant ones in Dylan samples. Transcript analysis indicated that related genes exhibited different expression trends; the heat map also revealed the different behaviors of the hydrolysates of the proteins 1 and 2 nitrogen sources. The effects of nitrogenous fertilizers at the proteomic and agronomic levels revealed that plants fertilized with synthesis or rhizovit gave the best results concerning yield, whereas rhizovit and protein hydrolysates were most effective for proteins content in the grain (% of dry weight). Therefore, all parameters measured in this study indicated that different kinds of nitrogen fertilization used have a relevant impact on plant growth and production.

Identification by serological proteome analysis of paramyosin as prominent allergen in dust mite allergy.

Component-resolved diagnosis (CRD) of IgE-mediated hypersensitivities is challenged by the possibility that single patients are sensitized to components not commercially available to the clinical lab. Here, we studied a patient with positive extract-based diagnosis of house dust mite (HDM) allergy based on routine in vivo (prick test) and in vitro (serum specific IgE) tests, whose serum scored negative for IgE to the three recombinant allergens routinely used in CRD (group 1 allergens, group 2 allergens and tropomyosin). By means of serological proteome analysis via two-dimensional gel electrophoresis combined with immunoblotting and mass spectrometry, paramyosin (group 11 allergen: Der f 11 and Der p 11) was identified as the allergen component recognized by serum IgE from this patient in a raw allergen extract. Nine patients (64%) had IgE to Der p 11 in a group of 14 HDM allergic patients. BIOLOGICAL SIGNIFICANCE: Our results add up to previous reports indicating that paramyosin is a clinically relevant HDM allergen and highlight that it can represent, in some patients, the first sensitizing component of this allergen source. This suggests that, at the moment, the use of allergen extract for the purpose of measuring IgE reactivity cannot be replaced by component resolved diagnosis and that group 11 allergens should be included among allergen components routinely tested in the clinical laboratory.

Serological immune response against ADAM10 pro-domain is associated with favourable prognosis in stage III colorectal cancer patients.

A humoral immune response against aberrant tumor proteins can be elicited in cancer patients, resulting in the production of auto-antibodies (Abs). By serological proteome analysis we identified the surface membrane protein ADAM10, a metalloproteinase that has a role in epithelial-tumor progression and invasion, as a target of the immune response in colorectal cancer (Crc). A screening carried out on the purified protein using testing cohorts of sera (Crc patients n = 57; control subjects n = 39) and validation cohorts of sera (Crc patients n = 49; control subjects n = 52) indicated that anti-ADAM10 auto-Abs were significantly induced in a large group (74%) of colon cancer patients, in particular in patients at stage II and III of the disease. Interestingly, in Crc patients classified as stage III disease, the presence of anti-ADAM10 auto-Abs in the sera was associated with a favourable follow-up with a significant shifting of the recurrence-free survival median time from 23 to 55 months. Even though the ADAM10 protein was expressed in Crc regardless the presence of auto-Abs, the immature/non-functional isoform of ADAM10 was highly expressed in the tumor of anti-ADAM10-positive patients and was the isoform targeted by the auto-Abs. In conclusion, the presence of anti-ADAM10 auto-Abs seems to reflect the increased tumor expression of the immunogenic immature-ADAM10 in a group of Crc patients, and is associated with a favourable prognosis in patients at stage III of the disease.

Natural Antibodies to Tumor-Associated Antigens.

Natural autoantibodies raised by humoral immune response to cancer can be exploited to identify potential tumor-associated antigens (TAAs), and might constitute new putative prognostic and/or diagnostic biomarkers. Here we describe how sera from tumor patients can be used to identify TAAs by screening antibody immunoreactivity against the cancer proteome resolved by two-dimensional gel electrophoresis.

Protein disulfide isomerase A3-specific Th1 effector cells infiltrate colon cancer tissue of patients with circulating anti-protein disulfide isomerase A3 autoantibodies.

To investigate novel colorectal cancer (CRC)-associated antigens that could be targets of humoral or cellular responses, we analyzed the reactivity of serum from a long-surviving CRC patient (for more than 100 months of follow-up) in clinical remission, by serologic proteome analysis. Two-dimensional Western blotting (2D-WB) and mass spectrometry analysis revealed a strong reactivity of this serum against protein disulfide isomerase A3 (PDIA3). Anti-PDIA3 antibodies are not a diagnostic marker of CRC, 2D-WB and Luminex analysis revealed that they were equally present in about 10% of sera from healthy subjects and CRC patients. Kaplan-Meier analysis of survival in CRC patient cohort, after 48 months of follow-up, showed a trend of higher survival in patients with increased levels of autoantibodies to PDIA3. Therefore, the interplay between the presence of these antibodies and T-cell response was investigated. Peripheral blood T cells from CRC patients with high immunoglobulin G (IgG) reactivity to PDIA3 also secreted interferon gamma (IFN-γ) when stimulated in vitro with recombinant PDIA3, whereas those from CRC with low IgG reactivity to PDIA3 did not. PDIA3-pulsed dendritic cells efficiently induced proliferation and IFN-γ production of autologous CD4(+) and CD8(+) T cells. Finally, ex vivo analysis of tumor-infiltrating T lymphocytes from CRC patients with autoantibodies to PDIA3 revealed that PDIA3-specific Th1 effector cells accumulated in tumor tissue. These data indicate that the presence of autoantibodies to PDIA3 favors the development of an efficient and specific T-cell response against PDIA3 in CRC patients. These results may be relevant for the design of novel immunotherapeutic strategies in CRC patients.

Image Pretreatment Tools I: Algorithms for Map Denoising and Background Subtraction Methods.

One of the critical steps in two-dimensional electrophoresis (2-DE) image pre-processing is the denoising, that might aggressively affect either spot detection or pixel-based methods. The Median Modified Wiener Filter (MMWF), a new nonlinear adaptive spatial filter, resulted to be a good denoising approach to use in practice with 2-DE. MMWF is suitable for global denoising, and contemporary for the removal of spikes and Gaussian noise, being its best setting invariant on the type of noise. The second critical step rises because of the fact that 2-DE gel images may contain high levels of background, generated by the laboratory experimental procedures, that must be subtracted for accurate measurements of the proteomic optical density signals. Here we discuss an efficient mathematical method for background estimation, that is suitable to work even before the 2-DE image spot detection, and it is based on the 3D mathematical morphology (3DMM) theory.

Nonlinear Dimensionality Reduction by Minimum Curvilinearity for Unsupervised Discovery of Patterns in Multidimensional Proteomic Data.

Dimensionality reduction is largely and successfully employed for the visualization and discrimination of patterns, hidden in multidimensional proteomics datasets. Principal component analysis (PCA), which is the preferred approach for linear dimensionality reduction, may present serious limitations, in particular when samples are nonlinearly related, as often occurs in several two-dimensional electrophoresis (2-DE) datasets. An aggravating factor is that PCA robustness is impaired when the number of samples is small in comparison to the number of proteomic features, and this is the case in high-dimensional proteomic datasets, including 2-DE ones. Here, we describe the use of a nonlinear unsupervised learning machine for dimensionality reduction called minimum curvilinear embedding (MCE) that was successfully applied to different biological samples datasets. In particular, we provide an example where we directly compare MCE performance with that of PCA in disclosing neuropathic pain patterns, hidden in a multidimensional proteomic dataset.