New approach based on fuzzy logic and principal component analysis for the classification of two-dimensional maps in health and disease. Application to lymphomas

Two-dimensional (2D) electrophoresis is the most wide spread technique for the separation of proteins in biological systems. This technique produces 2D maps of high complexity, which creates difficulties in the comparison of different samples. The method proposed in this paper for the comparison of different 2D maps can be summarised in four steps: (a) digitalisation of the image; (b) fuzzyfication of the digitalised map in order to consider the variability of the two-dimensional electrophoretic separation; (c) decoding by principal component analysis of the previously obtained fuzzy maps, in order to reduce the system dimensionality; (d) classification analysis (linear discriminant analysis), in order to separate the samples contained in the dataset according to the classes present in said dataset. This method was applied to a dataset constituted by eight samples: four belonging to healthy human lymph-nodes and four deriving from non-Hodgkin lymphomas. The amount of fuzzyfication of the original map is governed by the sigma parameter. The larger the value, the more fuzzy theresulting transformed map. The effect of the fuzzyfication parameter was investigated, the optimal results being obtained for sigma = 1.75 and 2.25. Principal component analysis and linear discriminant analysis allowed the separation of the two classes of samples without any misclassification.     

Spot overlapping in two-dimensional polyacrylamide gel electrophoresis separations: a statistical study of complex protein maps

A statistical approach able to extract the information contained in a two-dimenisional polyacrylamide gel electrophoresis (2-D PAGE) separation is here reported. The method is based on the quantitative theory of peak overlapping, a procedure previously developed by the authors and here extended to 2-D separations. The whole map is divided into many strips in order to obtain 1-D separations on which the statistic procedure is applied: the developed algorithms, on the basis of spot experimental data (intensity and spatial coordinates) permit to estimate the intrinsic number of components and to single out the specific order present in spot positions. The procedure was validated on computer-simulated maps. Its applicability to real samples was tested on maps obtained from literature sources. The following important information on protein mixtures can be extracted: (i) the number of proteins can be accurately estimated, on the basis of the spatial coordinates and intensities of spots detected in the 2-D PAGE map; (ii) the model describing distribution of interdistance between adjacent spots can be identified in both the separation dimensions; (iii) the presence of repeated interdistances in spot positions in the maps can be easily singled out: these regularities suggest specific protein modifications.     

Decoding two-dimensional polyacrylamide gel electrophoresis complex maps by autocovariance function: a simplified approach useful for proteomics

This paper describes a mathematical approach applied for decoding the complex signal of two-dimensional polyacrylamide gel electrophoresis maps of protein mixtures. The method is helpful in extracting analytical information since separation of all the proteins present in the sample is still far from being achieved and co-migrating proteins are generally present in the same spot. The simplified method described is based on the study of the 2-D autocovariance function (2D-ACVF) computed on an experimental digitized map. The first part of the 2D-ACVF allows for the estimation of the number of proteins present in the sample (2D-ACVF computed at the origin) and of the separation performance (mean spot size). Moreover, the 2D-ACVF plot is a powerful tool in identifying order in the spot position, and singling it out from the complex separation pattern. This method was validated on synthetic maps obtained by computer simulation to describe 2-D PAGE real maps and reference maps retrieved from the SWISS-2DPAGE database. The results obtained are discussed by focusing on specific information relevant in proteomics: sample complexity, separation performance, and identification of spot trains related to post-translational modifications.     

Spot overlapping in two-dimensional polyacrylamide gel electrophoresis maps: relevance to proteomics

Proteomics requires a large-scale, simultaneous separation of proteins from a mixture, assessment of the relative abundance of these molecules, and identification and characterization of each component. In 2-D PAGE separations, the best method of choice for protein analysis, separation of all the proteins present in the sample is still far to be achieved and comigrating proteins in the same spot are in general present. A statistical estimation of the degree of spot overlapping present in a 2-D PAGE separation is here described: for different conditions of spot overcrowding in the map, the degree of overlapping can be quantified in terms of purity degree of each spot or percentage of proteins that will appear in the map as a single spot. A computer simulation approach is described: it is based on the protein separation pattern present in the experimental maps. The results thus obtained are compared to a theoretical model (statistical degree of peak overlapping model) based on random spot position. The described procedures were applied to an experimental reference map of human plasma. The severity of spot overlapping in 2-D PAGE maps is estimated and the influence of different experimental conditions (strip dimension, detector system performance, pI range) is discussed. These informations are useful to quantitatively estimate the degree of error associated with identification and quantitation of each protein and to set-up experimental conditions which will increase resolution and greatly decrease the probability of spot overlapping.     

A turning point in proteome analysis: sample prefractionation via multicompartment electrolyzers with isoelectric membranes

Sample prefractionation, as obtained via multicompartment electrolyzers with isoelectric membranes, greatly enhanced the load ability, resolution and detection sensitivity of two-dimensional (2-D) maps in proteome analysis. This was demonstrated with different samples. In an Escherichia coli total cell extract, analysis by a 2-D map run in a pH 4-5 gradient showed many more spots when prefractionated, as compared with standard maps available in databases such as SWISS-2DPAGE. Analysis of human plasma in the pH 3-6 range showed an increase in the number of highly acidic proteins in the fractionated sample compared to whole plasma. With both samples no protein precipitation or smears occurred and much larger sample amounts could be loaded upon prefractionation, so that a large number of spots could be visualized by Coomassie staining, which is fully compatible with subsequent matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis.     

Proteomic analysis of cellular soluble proteins from human bronchial smooth muscle cells by combining nondenaturing micro 2DE and quantitative LC‐MS/MS. 2. Similarity search between protein maps for the analysis of protein complexes

Human bronchial smooth muscle cell soluble proteins were analyzed by a combined method of nondenaturing micro 2DE, grid gel‐cutting, and quantitative LC‐MS/MS and a native protein map was prepared for each of the identified 4323 proteins [1]. A method to evaluate the degree of similarity between the protein maps was developed since we expected the proteins comprising a protein complex would be separated together under nondenaturing conditions. The following procedure was employed using Excel macros; (i) maps that have three or more squares with protein quantity data were selected (2328 maps), (ii) within each map, the quantity values of the squares were normalized setting the highest value to be 1.0, (iii) in comparing a map with another map, the smaller normalized quantity in two corresponding squares was taken and summed throughout the map to give an “overlap score,” (iv) each map was compared against all the 2328 maps and the largest overlap score, obtained when a map was compared with itself, was set to be 1.0 thus providing 2328 “overlap factors,” (v) step (iv) was repeated for all maps providing 2328 × 2328 matrix of overlap factors. From the matrix, protein pairs that showed overlap factors above 0.65 from both protein sides were selected (431 protein pairs). Each protein pair was searched in a database (UniProtKB) on complex formation and 301 protein pairs, which comprise 35 protein complexes, were found to be documented. These results demonstrated that native protein maps and their similarity search would enable simultaneous analysis of multiple protein complexes in cells.     

Capillary electrophoresis/tandem mass spectrometry for analysis of proteins from two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis

Capillary electrophoresis/time-of-flight mass spectrometry(CE/TOFMS) has been used for analysis of in-gel digests of protein spots excised from two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (2-D SDS-PAGE). An off-line purification and preconcentration procedure with a Zip Tip is used before CE/TOFMS analysis which allows for detection of protein spots with <1 picomole of material from 2-D gels. The off-line procedure provides sufficient purification for analysis while maintaining the quality of the CE separation. Using this procedure, several proteins from Coomassie Blue and zinc negatively stained gels are identified by the peptide maps generated and database searching. CE/TOF tandem mass spectrometry is used for the confirmation of database searching results and structural analysis of peptides that do not match the expected peptide maps obtained from the database in order to identify structural modifications. Several modifications were pinpointed and identified by this method.     

Proteomic profiling combining solution-phase isoelectric fractionation with two-dimensional gel electrophoresis using narrow-pH-range immobilized pH gradient gels with slightly overlapping pH ranges

This paper describes a simple new approach toward improving resolution of two-dimensional (2-D) protein gels used to explore the mammalian proteome. The method employs sample prefractionation using solution-phase isoelectric focusing (IEF) to split the mammalian proteome into well-resolved pools. As crude samples are thus prefractionated by pI range, very-narrow-pH-range 2-D gels can be subsequently employed for protein separation. Using custom pH partition membranes and commercially available immobilized pH gradient (IPG) strips, we maximized the total separation distance and throughput of seven samples obtained by prefractionation. Both protein loading capacity and separation quality were higher than the values obtained by separation of fractionated samples on narrow-pH-range 2-D gels; the total effective IEF separation distance was ~82 cm over the pH range pH 3–10. This improved method for analyzing prefractionated samples on narrow-pH-range 2-D gels allows high protein resolution without the use of large gels, resulting in decreased costs and run times.      

On graphical and numerical characterization of proteomics maps

We outlined a mathematical approach suitable for characterization of experimental data given by 2-D densitograms. In particular we consider numerical characterization of proteomics maps. The basis of our approach is to order "spots" of a 2-D map and assign them unique labels (that in general will depend on the criteria used for ordering). In this way a map is "translated" into a sequence. In the next step one associates with the generated sequence a geometrical path and views such a path as a mathematical object that needs characterization. We have ordered spots representing proteins in 2-D gel plates according to their relative intensities which results in a zigzag path that produces a complicated "fingerprint" pattern. Mathematical characterization of zigzag pattern follows similar mathematical characterizations of embedded patterns based on matrices, the elements of which are given as quotients of Euclidean distance between spots and the distance along the zigzag path. The leading eigenvalue of constructed matrices is taken to represent characterization of the original 2-D map. Comparison of different 2-D maps (simulated by using random generator) allows one to construct partial order, which although qualitative in nature gives some insight into perturbation induced by foreign agents to the proteome of the control cell.     

Quantitative and qualitative precision improvements by effective mobility-scale data transformation in capillary electrophoresis analysis

By transforming the time-based x-axis of electropherograms in capillary zone electrophoresis (CZE) into the corresponding effective mobility-scale, we propose a simple and robust data representation for a better qualitative and quantitative capillary electrophoresis (CE) analysis. The time scale of the raw electrophoretic data (detection signal versus time) is transformed into an effective electrophoretic mobility scale (mu eff-scale) with account of the electroosmotic flow (EOF) peak or of an internal standard of known effective mobility. With the new scaling (detection signals versus effective mobility), the obtained electropherograms are more representative of the velocity-based electrophoretic separation and the comparison of complete electropherograms is directly possible. This is of importance when tracking peaks in real samples where alteration in EOF stability can occur or when comparing electrophoretic runs from different experimental setups (independence in column length and voltage). Beside the qualitative possibilities, a quantitative improvement is achieved in the mu eff-scale with significant better peak area reproducibility and equal to more precision in quantitative analysis than with the primary time-scale integration.     

An innovative method for quality control of conjugated Haemophilus influenzae vaccines: A short review of two-dimensional nanoparticle electrophoresis

This article provides an overview of a 2D agarose electrophoretic procedure for the characterization of semi-synthetic Haemophilus influenzae type b meningitis vaccines that were prepared for the immunization of small children. The analysis of such vaccines has been particularly challenging because the vaccine particles (i) are highly negatively charged, (ii) are as large as or even larger than intact viruses, and (iii) have a continuous (polydisperse) size distribution because of randomizing steps in the vaccine production (sonification and crosslinking). As a result of these characteristics, 1D electrophoresis of the vaccines produced smears without discernable peaks, but with a second dimension of separation a characteristic vaccine fingerprint was obtained. Whereas O’Farrell gels can accomplish a 2D separation according to size and charge for samples with protein-sized particles, nondenaturing 2D agarose electrophoresis achieves a similar result for much larger virus-sized particles. The separation principle, however, is different. Even though the 2D electrophoretic method was developed from 1983 to 1995, it remains a promising tool for vaccine quality control and for predicting vaccine effectiveness. Modern technology makes the analysis significantly more practical and affordable than it was more than 10 years ago, and the method is applicable to a variety of conjugated vaccines and complex mixtures of virus-sized particles.     

Two-dimensional strong cation-exchange liquid chromatography/reversed-phase pressurized capillary electrochromatography for separation of complex samples

A 2-D separation platform was constructed using micro strong cation-exchange liquid chromatography (μ-SCXLC) and reversed-phase pressurized capillary electrochromatography (RP-pCEC) for the analysis of complex samples. Samples were fractionated by the first-dimension μ-SCXLC with a linear solvent gradient and then injected into the second-dimension RP-pCEC for further separation. The μ-SCXLC/RP-pCEC 2-D system with three separation mechanisms, namely strong cation-exchange, reversed-phase chromatography and electrophoresis, provided high selectivity, high resolution and high peak capacity compared to one-dimensional chromatographic approaches. Separation effectiveness of this 2-D system was demonstrated by the analysis of different kinds of complex samples, such as traditional Chinese medicine Cortex Phellodendri, bovine serum albumin (BSA) tryptic digest and real serum tryptic digest. A theoretical peak capacity of approximately 1200 was achieved, which proves its promising potential for the separation and analysis of complex samples.     

A mouse serum two-dimensional gel map: application to profiling burn injury and infection

With the importance of mouse as a model to study human diseases and the human and rat plasma/serum two-dimensional (2-D) maps being extensively annotated, this study was aimed at constructing a detailed mouse serum 2-D map. Serum proteins from two different inbred strains of mice (BALB/cJ and C57BL/6J) and mice subjected to two different inflammatory stimuli (20% burn injury and lipopolysaccharide (LPS) injection) were separated on overlapping gels covering pH 3-8 and stained with SYPRO Ruby dye. The tryptic peptides from the resolved spots were analyzed by mass spectrometry, leading to the identification of 38 different gene products. With the exception of major urinary proteins found in abundance in male C57BL/6J mice, little strain difference of the mouse serum 2-D was observed. Many proteins detected in the mouse serum 2-D map were not reported in human or rat serum 2-D maps including epidermal growth factor receptor. Three major murine acute-phase proteins (APPs), haptoglobin, serum amyloid A, and serum amyloid P, were highly induced by both inflammatory stimuli. Image analysis shows that the variations of APPs between these two inflammatory models were not uniform although LPS (100 microg/animal) in general was more effective than 20% burn injury in inducing APPs. Serum amyloid A, much more sensitive to endotoxin than burn injury, may represent a sensitive marker to differentiate these two different inflammatory states.     

Microchip analysis of plant glucosinolates

We describe a new and selective analytical method for the separation and quantitation of plant glucosinolates. The new method, which utilizes microchip CE (micro-CE) with fluorescence detection, circumvents the multistep procedures characteristic of conventional methods. Glucosinolates form charge transfer complexes with the xanthene dyes phloxine-B and eosin-B. The glucosinolates-phloxine-B complex cannot be excited at 470 nm. Thus, the decrease in peak intensity of phloxine-B after complex formation is used to quantitatively measure total glucosinolates in Arabidopsis thaliana seeds. For qualitative analysis, complex formation with eosin-B is used. The sensitivity of eosin-B detection at excitation/emission 470 nm/540 nm was low. However, sensitivity increased following complex formation with sinigrin (> or =3 microg/mL). A batch-learning, self-organizing map was applied to visualize and organize analytical data into 2-D matrix with similar and related data clustered together or near each other. This organized matrix was used to optimize electrophoretic conditions for the analysis. This study suggests potential applications of micro-CE in plant metabolomics analyses without use of labeling fluorophores.     

t检验法用于评价中药色谱指纹图谱的相似度

本文采用t检验法评价中药色谱指纹图谱的相似度。利用来自六个不同厂家的三黄片样品的高效液相色谱指纹图谱数据来验证t检验法的适用性,并用主成分分析(PCA)进一步验证方法的可行性。在PCA的聚类图上,t检验无显著性差异的样品聚为一类,t检验有显著性差异的样品各为一类。结果表明:用t检验法能客观地反映中药样品间的相似性和差异性。     

Study of the similarity between distribution maps of concentration in near-infrared spectroscopy chemical imaging obtained by different multivariate calibration approaches

Nowadays, near-infrared spectroscopy chemical imaging (NIR-CI) has been widely used in pharmaceutical analysis since it provides important surface information about the samples. In this work the information of NIR-CI at the pixel level was compared through calculation of the similarity between distribution maps of concentration obtained by different multivariate calibration approaches. The comparison was performed by using four different multivariate methods (MCR, MLR, CLS and PLS) in analysis of carbamazepine pharmaceutical formulations. For global determination, all models developed showed RMSEP below 1.9% (w/w) for active principal ingredient (API) and better than 4.6% (w/w) for excipients. Also, the distribution maps obtained by PLS, CLS and MCR showed great similarity for all compounds of the formulation as well with concentrations in the tablets. However, comparing the distribution maps obtained by MLR with those from the other chemometric tools, a lower similarity was observed. Thus, this fitted model does not ensure, by itself, that the images obtained are reliable or accurate. The paper also compares the distribution maps of concentrations obtained from all constituents present in the pharmaceutical formulation with their respective micrographs.     

Proteomic analysis of rat brain tissue: comparison of protocols for two-dimensional gel electrophoresis analysis based on different solubilizing agents

The present study reports a comparison of recently described solubilizing methods, to set up a simple protocol for obtaining two-dimensional (2-D) gel electrophoresis maps of brain tissue. Different protocols were used for preparing rat brain homogenates and the resulting maps were compared by image analysis. Three different detergents, two delipidation methods, and introduction of a fractionation step based on different protein solubility in surfactants, were evaluated. When using efficient zwitterionic detergents (3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonate, CHAPS; amidosulfobetaine 14, ASB-14), the patterns obtained by direct loading of total extracts were qualitatively overlapping with patterns obtained from fractionated samples. In contrast, a weaker nonionic agent (Nonidet P-40, NP-40) produced a different protein pattern in the collected fractions. Delipidation did not improve the results for all the different extraction methods. Immunoblots performed with antibodies recognizing cytosolic and membrane-spanning proteins, which were detected as nondegraded spots, showed that membrane proteins with intermediate molecular mass could be recovered. We suggest, as a simple and efficient method for preparing rat brain maps, the homogenization in a solution containing an efficient zwitterionic surfactant, which allows to solubilize cytosolic and membrane proteins in a single step. Alternatively, a fractionation can be carried out on samples homogenized by a weak solubilizing agent, a more labor-intensive effort resulting in a larger number of proteins on two maps.     

Proteome analysis of human plasma and amniotic fluid by Off-Gel isoelectric focusing followed by nano-LC-MS/MS

This paper presents a comparative proteomic analysis of human maternal plasma and amniotic fluid (AF) samples from the same patient at term of pregnancy in order to find specific AF proteins as markers of premature rupture of membranes, a complication frequently observed during pregnancy. Maternal plasma and the corresponding AF were immunodepleted in order to remove the six most abundant proteins before the systematic analysis of their protein composition. The protein samples were then fractionated by IEF Off-Gel electrophoresis (OGE), digested and analyzed with nano-LC-MS/MS separation, revealing a total of 73 and 69 proteins identified in maternal plasma and AF samples, respectively. The proteins identified in AF have been compared to those identified in the mother plasma as well as to the reference human plasma protein list reported by Anderson et al. (Mol. Cell. Proteomics 2004, 3, 311-326). This comparison showed that 26 proteins were exclusively present in AF and not in plasma among which 10 have already been described to be placenta or pregnancy specific. As a further validation of the method, plasma proteins fractionated by OGE and analysed by nano-LC-MS/MS have been compared to the Swiss 2-D PAGE reference map by reconstructing a map that matches 2-D gel and OGE experimental data. This representation shows that 36 of 49 reference proteins could be identified in both data sets, and that isoform shifts in pI are well conserved in the OGE data sets.     

Synthesis, analytical characterization and initial capillary electrophoretic use in an acidic background electrolyte of a new, single-isomer chiral resolving agent: heptakis(2-O-sulfo-3-O-methyl-6-O-acetyl)-β-cyclodextrin

The sodium salt of heptakis(2-O-sulfo-3-O-methyl-6-O-acetyl)cyclomaltoheptaose (HAMS), the first single-isomer sulfated β-CD that carries the sulfo group exclusively at the C2 position, has been synthesized. The purity of each synthetic intermediate and of the final product was determined by hydrophilic interaction (HILIC) and reversed-phase HPLC. The structural identity of each intermediate and of the final product was verified by 1-D and 2-D NMR spectroscopy and MALDI-TOF MS. HAMS was used for the capillary electrophoretic separation of the enantiomers of a set of non-ionic and weak base analytes in pH 2.5 background electrolytes. Rapid separations with satisfactory peak resolution values were obtained for most enantiomers using low concentrations of HAMS. The effective mobilities and separation selectivities were dependent on the concentration of HAMS according to the predictions of the charged resolving agent migration model. The separation selectivities observed with HAMS, heptakis(2-O-methyl-3-O-acetyl-6-O-sulfo)cyclomaltoheptaose and heptakis(2-O-methyl-3,6-di-O-sulfo)cyclomaltoheptaose were different for some of the analytes studied in detail.     

Toward high sequence coverage of proteins in human breast cancer cells using on-line monolith-based HPLC-ESI-TOF MS compared to CE MS

A method is developed toward high sequence coverage of proteins isolated from human breast cancer MCF10 cell lines using a 2-D liquid separations. Monolithic-capillary columns prepared by copolymerizing styrene with divinylbenzene are used to achieve high-resolution separation of peptides from protein digests. This separation is performed with minimal sample preparation directly from the 2-D liquid fractionation of the cell lysate. The monolithic column separation is directly interfaced to ESI-TOF MS to obtain a peptide map. The protein digests were also analyzed by MALDI-TOF MS and an accurate M(r) of the intact protein was obtained using an HPLC-ESI-TOF MS. The result is that these techniques provide complementary information where nearly complete sequence coverage of the protein is obtained and can be compared to the experimental M(r) value. The high sequence coverage provides information on isoforms and other post-translational modifications that would not be available from methods that result in low sequence coverage. The results from the use of monolithic columns are compared to that obtained by CE-MS. The monolithic column separations provide a rugged and highly reproducible method for separating protein digests prior to MS analysis and is suited to confidently identify biomarkers associated with cancer progression.