Journal of Chromatography B (v.833, #1)

Preface by Lello Zolla; Pier Giorgio Righetti (1-2).

Alzheimer's disease is a progressive neurodegenerative disease associated with loss of memory and cognition. One hallmark of AD is the accumulation of amyloid β-peptide (Aβ), which invokes a cascade of oxidative damage to neurons that can eventually result in neuronal death. Several markers of oxidative stress have been identified in AD brain, thus providing greater understanding into potential mechanisms involved in the disease pathogenesis and progression. In the present article, we review the application of redox proteomics to the identification of oxidized proteins in AD brain and also our recent findings on amyloid β-peptide (Aβ)-associated in vivo and in vitro models of AD. Our redox proteomics approach has made possible the identification of specifically oxidized proteins in Alzheimer's disease (AD) brain, providing for the first time evidence on how oxidative stress plays a crucial role in AD-related neurodegeneration. The information obtained has great potential to aid in determining the molecular pathogenesis in and detecting disease markers of AD, as well as identifying potential targets for drug therapy in AD. Application of redox proteomics to study cellular events, especially related to disease dysfunction, may provide an efficient tool to understand the main mechanisms involved in the pathogenesis and progression of oxidative stress-related neurodegenerative disorders.
Keywords: Redox proteomics; Alzheimer's disease; Oxidative stress; Protein oxidation;

Purification and characterization of phycocyanin from the blue-green alga Aphanizomenon flos-aquae by Serena Benedetti; Sara Rinalducci; Francesca Benvenuti; Sonia Francogli; Silvia Pagliarani; Luca Giorgi; Mauro Micheloni; Gian Maria D’Amici; Lello Zolla; Franco Canestrari (12-18).
Aphanizomenon flos-aquae (AFA) is a blue-green alga and represents a nutrient-dense food source. In this study the presence of phycocyanin (PC), a blue protein belonging to the photosynthetic apparatus, has been demonstrated in AFA. An efficient method for its separation has been set up: PC can be purified by a simple single step chromatographic run using a hydroxyapatite column (ratio A 620/A 280 of 4.78), allowing its usage for health-enhancing properties while eliminating other aspecific algal components. Proteomic investigation and HPLC analysis of purified AFA phycobilisomes revealed that, contrary to the well-characterized Synechocystis and Spirulina spp., only one type of biliprotein is present in phycobilisomes: phycocyanins with no allo-phycocyanins. Two subunit polypeptides of PC were also separated: the β subunit containing two bilins as chromophore and the α subunit containing only one.
Keywords: Aphanizomenon flos-aquae; Cyanobacteria; Phycocyanin; Phycocyanobilin; Purification;

Quasi-isoelectric buffers for protein analysis in a fast alternative to conventional capillary zone electrophoresis by Paolo Antonioli; Martha E. Mendieta; Roberto Sebastiano; Attilio Citterio; Gabriel Peltre; Jean-Marc Busnel; Stephanie Descroix; Giovanni Candiano; Pier Giorgio Righetti (19-25).
Two different approaches are here reported for obtaining ultra-narrow pI cuts from 2-pH unit wide carrier ampholyte ranges, as commercially available, for use as quasi-isoelectric buffers in capillary electrophoresis separations of proteins. One of them uses multicompartment electrolyzers endowed with isoelectric membranes (Immobiline technology); the other employs the Rotofor equipment. Although the first approach results in more precise pI cuts, the latter technique is much faster, easier to handle and permits the immediate collection of 20 fractions in a single run. This results in ultra-narrow, ca. 0.1-pH unit intervals, uniformly spaced apart along the original wider gradient utilized for the fractionation. It is here shown that such quasi-isoelectric buffers, especially those in the pH 8–9 interval, have the unique property of coating the silica wall, thus preventing interaction of the proteins with the silica surface, that would otherwise totally disrupt the separation. On the contrary, such a shielding is not obtained in control, non isoelectric buffers (such as phosphate), that give very poor separations in uncoated capillaries. It is hypothesized that such a unique shielding effect is due to the oligo-amino backbone of the carrier ampholytes, typically composed (in the Vesterberg's synthetic approach) of 4–6 nitrogens spaced apart by ethylene moieties. Although such oligoprotic buffers should bear, in the isoelectric state, just one positive and one negative charge, they might be transiently ionized upon contact with the silanols, thus inducing a cooperative binding to the silica wall.
Keywords: Ultra-narrow-pH ampholytes cuts; Fractionation techniques; Isoelectric buffer; Uncoated-capillary CZE;

Identification and characterization of digestive serine proteases from inhibitor-resistant Helicoverpa zea larval midgut by Mariateresa Volpicella; Jan Cordewener; Maarten A. Jongsma; Raffaele Gallerani; Luigi R. Ceci; Jules Beekwilder (26-32).
Protease inhibitors mediate a natural form of plant defence against insects, by interfering with the digestive system of the insect. In this paper, affinity chromatography was used to isolate trypsins and chymotrypsins from Helicoverpa zea larvae, which had been raised on inhibitor-containing diet. Sensitivity of the fractions to inhibition by plant proteinase inhibitors was tested, and compared to the sensitivity of proteinases found in insects raised on diet to which no inhibitor had been added. The isolated chymotrypsin activity was found to be less sensitive to plant protease inhibitors. The sensitivity of the isolated trypsin activity was found to be intermediate between completely sensitive trypsins and completely insensitive forms that have been previously described. Mass spectrometry was used to identify one trypsin and two chymotrypsins in the partially purified protease fraction. The sequence features of these proteases are discussed in relation to their sensitivity to inhibitors. The results provide insight in the enzymes deployed by Helicoverpa larvae to overcome plant defence.
Keywords: Affinity chromatography; Gut; Helicoverpa zea; Protease inhibitor; Trypsin;

Reducing protein concentration range of biological samples using solid-phase ligand libraries by Luc Guerrier; Vanitha Thulasiraman; Annalisa Castagna; Frederic Fortis; Shanhua Lin; Lee Lomas; Pier Giorgio Righetti; Egisto Boschetti (33-40).
The discovery of specific polypeptides of diagnostic relevance from a biological liquid is complicated by the overall vast number and the large concentration range of all polypeptides/proteins in the sample. Depletion or fractionation methodologies have been used for selectively removing abundant proteins; however, they failed to significantly enrich trace proteins. Here we expand upon a new method that allows the reduction of the protein concentration range within a complex mixture, like neat serum, through the simultaneous dilution of high abundance proteins and the concentration of low abundance ones in a single, simple step. This methodology utilizes solid-phase ligand libraries of large diversity. With a controlled sample-to-ligand ratio it is possible to modulate the relative concentration of proteins such that a large number of peptides or proteins that are normally not detectable by classical analytical methods become, easily detectable. Application of this method for reducing the dynamic range of unfractionated serum is specifically described along with treatment of other biological extracts. Analytical surface enhanced laser desorption/ionization mass spectrometry (SELDI-MS) technology and mono- and two-dimensional electrophoresis (1-DE and 2-DE) demonstrate the increase in the number of proteins detected. Examples linking this approach with additional fractionation methods demonstrate a further increase in the number of detectable species using either the so-called “top down” or “bottom up” approaches for proteomics analysis. By enabling the detection of a greater proportion of polypeptides/proteins within a sample, this method may contribute significantly towards the discovery of new biomarkers of diagnostic relevance.
Keywords: Proteomics; Ligand library; Selective capture; Fractionation; Mass spectrometry; Electrophoresis; Dynamic range;

Differential proteomic analysis of nuclear extracts from thyroid cell lines by Anna Maria Salzano; Igor Paron; Alex Pines; Angela Bachi; Fabio Talamo; Nicoletta Bivi; Carlo Vascotto; Giuseppe Damante; Franco Quadrifoglio; Andrea Scaloni; Gianluca Tell (41-50).
Nuclear proteins play a major role in controlling cell functions. Differential proteomic analysis of nuclear proteins by combined 2D gel electrophoresis (2D-E) and mass spectrometry procedures can provide useful information to understand the control of cell proliferation and differentiation. To identify proteins involved in dedifferentiation, we used a differential proteomics approach by comparing nuclear extracts from the differentiated rat thyroid cell line FRTL-5 and the derived undifferentiated Ki-mol cell line, obtained by transformation with the Ki-ras oncogene. Thirteen proteins were identified as differently expressed in the nuclear compartment between the two cell lines. RT-PCR analysis performed on seven differently expressed genes showed that only in two cases the difference may be ascribable to a transcriptional mechanism. Since one of the identified proteins, namely apurinic apyrimidinic endonuclease/redox effector factor-1 (APE1/Ref-1), is suspected to play a role in thyroid tumorigenesis, we used a glutathione S-transferase (GST)-pulldown assay coupled to a 2D electrophoretic/matrix assisted laser desorption ionization-time of flight (MALDI-TOF)–mass spectrometry (MS) analysis to detect and identify its interacting partners. We show here that β-actin directly interacted with APE1/Ref-1, as confirmed by co-immunoprecipitation assays and that this interaction was enhanced by oxidative stress on FRTL-5 cells.
Keywords: Differential proteomics; Thyroid; Differentiation; APE1/Ref-1; Transcriptional regulation; Nucleus;

Decoding 2D-PAGE complex maps: Relevance to proteomics by Maria Chiara Pietrogrande; Nicola Marchetti; Francesco Dondi; Pier Giorgio Righetti (51-62).
This review describes two mathematical approaches useful for decoding the complex signal of 2D-PAGE maps of protein mixtures. These methods are helpful for interpreting the large amount of data of each 2D-PAGE map by extracting all the analytical information hidden therein by spot overlapping. Here the basic theory and application to 2D-PAGE maps are reviewed: the means for extracting information from the experimental data and their relevance to proteomics are discussed. One method is based on the quantitative theory of statistical model of peak overlapping (SMO) using the spot experimental data (intensity and spatial coordinates). The second method is based on the study of the 2D-autocovariance function (2D-ACVF) computed on the experimental digitised map. They are two independent methods that are able to extract equal and complementary information from the 2D-PAGE map. Both methods permit to obtain fundamental information on the sample complexity and the separation performance and to single out ordered patterns present in spot positions: the availability of two independent procedures to compute the same separation parameters is a powerful tool to estimate the reliability of the obtained results. The SMO procedure is an unique tool to quantitatively estimate the degree of spot overlapping present in the map, while the 2D-ACVF method is particularly powerful in simply singling out the presence of order in the spot position from the complexity of the whole 2D map, i.e., spot trains. The procedures were validated by extensive numerical computation on computer-generated maps describing experimental 2D-PAGE gels of protein mixtures. Their applicability to real samples was tested on reference maps obtained from literature sources. The review describes the most relevant information for proteomics: sample complexity, separation performance, overlapping extent, identification of spot trains related to post-translational modifications (PTMs).
Keywords: Two-dimensional maps; Spot overlapping; Bioinformatics; Chemometric methods;

Comparative proteomics and immunoproteomics of Helicobacter pylori related to different gastric pathologies by Roberta Mini; Giulia Bernardini; Anna Maria Salzano; Giovanni Renzone; Andrea Scaloni; Natale Figura; Annalisa Santucci (63-79).
Helicobacter pylori is a Gram-negative bacterium which causes ulcer, atrophic gastritis, adenocarcinoma, or mucosa-associated lymphoid tissue lymphoma. A comparative proteomic and immunoproteomic analysis was chosen to identify the antigenic patterns of three different H. pylori strains. These strains were probed against single sera from H. pylori-positive patients affected by gastric adenocarcinoma or duodenal ulcer. We found a quite heterogeneous antigenic pattern, both from strain and sera points of view, thus underlying both a strain- and a host-specificity. The different antigenic repertoires introduced the importance of the strain to be used for immunoblotting as a diagnostic test.
Keywords: Immunoproteomic;

Proteomic studies on the white matter of human brain by Stefano Pozzi Mucelli; Federico Odreman; Marlen Lujardo Gonzales; Ernestina Gerardi; Giorgio Stanta; Alessandro Vindigni (80-90).
Limited information on the protein expression profiles of the different components of mammalian brain is available to date. In the present study, proteomic analysis was performed on 32 white matter samples obtained from 8 different regions of brains of four post mortem cases. Proteins were separated by 2D gel electrophoresis and identified by mass spectrometry. Most of the protein spots (98%) are reproducibly present in all the samples analyzed. A total of 64 different proteins were identified and divided into seven functional groups. These include metabolic proteins (33%), structural proteins (9%), proteins involved in signal transduction (9%), blood proteins (8%), stress related proteins (23%), and proteins involved in the ubiquitin mediated proteolysis (6%). This protein database obtained from the white matter of human brain contributes to deepen our knowledge on the molecular mechanisms that control several pathologies affecting this key component of the brain.
Keywords: Proteomics; Human brain; Brain white matter; 2D electrophoresis; Mass spectrometry;

A 2-D liquid-phase chromatography for proteomic analysis in plant tissues by Andrea Pirondini; Giovanna Visioli; Aliosha Malcevschi; Nelson Marmiroli (91-100).
Two-dimensional liquid chromatography based on a high-performance chromatofocusing in the first dimension followed by high-resolution reversed-phase chromatography in the second dimension can be used as a complementary approach to protein separation with two-dimensional gel electrophoresis. In this work, Arabidopsis thaliana proteins obtained from different tissue extracts were resolved by using a new automated system, ProteomeLab PF 2D commercialized by Beckman Coulter (Fullerton, CA, USA). In particular, protein patterns obtained after two different extraction procedures (MgSO4 and urea buffer) were compared. Reproducibility of the protein patterns was also confirmed in different injections of the same sample and in the comparative analyses of some proteins by MALDI-TOF/MS. Computer analysis of the chromatograms revealed that with this two-dimensional liquid phase technique, hundreds of “virtual bands” can be identified and compared in crude plant protein lysates.
Keywords: Arabidopsis thaliana; Comparative proteomics; Two-dimensional liquid chromatography; Protein crude extracts;

Proteomic study of muscle sarcoplasmic proteins using AUT-PAGE/SDS-PAGE as two-dimensional gel electrophoresis by Gianluca Picariello; Alessandra De Martino; Gianfranco Mamone; Pasquale Ferranti; Francesco Addeo; Michele Faccia; Salvatore SpagnaMusso; Aldo Di Luccia (101-108).
In the present study, an alternative procedure for two-dimensional (2D) electrophoretic analysis in proteomic investigation of the most represented basic muscle water-soluble proteins is suggested. Our method consists of Acetic acid-Urea-Triton polyacrylamide gel (AUT-PAGE) analysis in the first dimension and standard sodium dodecyl sulphate polyacrylamide gel (SDS-PAGE) in the second dimension. Although standard two-dimensional Immobilized pH Gradient-Sodium Dodecyl-Sulphate (2D IPG-SDS) gel electrophoresis has been successfully used to study these proteins, most of the water-soluble proteins are spread on the alkaline part of the 2D map and are poorly focused. Furthermore, the similarity in their molecular weights impairs resolution of the classical approach. The addition of Triton X-100, a non-ionic detergent, into the gel induces a differential electrophoretic mobility of proteins as a result of the formation of mixed micelles between the detergent and the hydrophobic moieties of polypeptides, separating basic proteins with a criterion similar to reversed phase chromatography based on their hydrophobicity. The acid pH induces positive net charges, increasing with the isoelectric point of proteins, thus allowing enhanced resolution in the separation. By using 2D AUT-PAGE/SDS electrophoresis approach to separate water-soluble proteins from fresh pork and from dry-cured products, we could spread proteins over a greater area, achieving a greater resolution than that obtained by IPG in the pH range 3–10 and 6–11. Sarcoplasmic proteins undergoing proteolysis during the ripening of products were identified by Matrix Assisted Laser Desorption/Ionization–Time of Flight (MALDI-ToF) mass spectrometry peptide mass fingerprinting in a easier and more effective way. Two-dimensional AUT-PAGE/SDS electrophoresis has allowed to simplify separation of sarcoplasmic protein mixtures making this technique suitable in the defining of quality of dry-cured pork products by immediate comparison of 2D maps to define the events occurring during their ripening and individuate candidate molecular markers of the characteristic proteolytic processes. Considering that, essentially, muscle endogenous enzymic activity, calpains and cathepsins, occur in the ripening process of dry-cured ham, whereas a combined action between endogenous and microbial enzymes takes place in the case of sausage ripening, these results provide deeper insight into the respective role of endogenous and microbial enzymes in performing proteolysis. Finally, image analysis and creation of data bank could be achieved to quickly identify and protect typical products.
Keywords: Two-dimensional AUT-PAGE/SDS-PAGE; MALDI-ToF fingerprinting; Sarcoplasmic proteins;

Determination of anti-HIV drug concentration in human plasma by MALDI-TOF/TOF by Stefania Notari; Carmine Mancone; Marco Tripodi; Pasquale Narciso; Mauro Fasano; Paolo Ascenzi (109-116).
The antiretroviral therapeutic drug monitoring is becoming increased in clinical care to determine the best dosage regimen adapted to each patient. Here, the determination of the anti-HIV drugs lamivudine, lopinavir, and ritonavir concentration in the plasma of HIV-infected patients by MALDI-TOF/TOF is reported. The volume of the plasma sample was 600 μL. Plasma samples were extracted by solid-phase (divinylbenzene and N-vinylpyrrolidone) and evaporated in a water bath under a nitrogen stream. The extracted samples were reconstituted with methanol (100 μL), mixed (1:1) with a saturated matrix solution (4-hydroxybenzoic acid in 50% acetonitrile–0.1% trifluoracetic acid), and spotted onto the MALDI-TOF/TOF sample target plate. The lamivudine, lopinavir and ritonavir concentration was determined by standard additions analysis. Regression of standard additions was linear over the anti-HIV drug concentration ranges explored (lamivudine, 0.010–1.0 pmol/μL; lopinavir and ritonavir, 0.0025–0.50 pmol/μL). Moreover, emtricitabine (i.e., the fluorinated analog of lamivudine) was used as the internal standard to determine the lamivudine concentration. The calibration curve was linear on the emtricitabine concentration ranging between 0.050 and 5.0 pmol/μL. The absolute recovery ranged between 80 and 110%. Values of the lamivudine, lopinavir and ritonavir concentration determined by MALDI-TOF/TOF are in excellent agreement with those obtained by HPLC-UV and HPLC-MS/MS. MALDI-TOF/TOF experiments allowed also the detection of the ritonavir metabolite R5. Zidovudine was undetectable by MALDI-TOF/TOF analysis because also the minimal laser intensity may induce the anti-HIV drug photolysis. The MALDI-TOF/TOF technique is useful to determine very low concentrations of anti-HIV drugs (0.0025–0.010 pmol/μL).
Keywords: Anti-HIV drug determination; Human plasma; MALDI-TOF/TOF;

Assessment of population structure by single nucleotide polymorphisms (SNPs) in goat breeds by L. Pariset; I. Cappuccio; P. Ajmone Marsan; S. Dunner; G. Luikart; P.R. England; G. Obexer-Ruff; C. Peter; D. Marletta; F. Pilla; A. Valentini (117-120).
Single nucleotide polymorphisms (SNPs) may be used in biodiversity studies and commercial tasks like traceability, paternity testing and selection for suitable genotypes. Twenty-seven SNPs were characterized and genotyped on 250 individuals belonging to eight Italian goat breeds. Multilocus genotype data were used to infer population structure and assign individuals to populations. To estimate the number of groups (K) to test in population structure analysis we used likelihood values and variance of the bootstrap samples, deriving optimal K from a drop in the likelihood and a rise in the variance plots against K.
Keywords: SNPs; Goat; Population structure;