ProteomeLab PF2D Research Articles

Identification of a Novel ITGαvβ6-Binding Peptide Using Protein Separation and Phage Display.

Purpose: Targeted therapies are regarded as promising approaches to increase 5-year survival rate of head and neck squamous cell carcinoma (HNSCC) patients.Experimental design: For the selection of carcinoma-specific peptides membrane proteome of HNO97 tumor cells fractionated by the ProteomeLab PF2D system and corresponding HNO97 cells were deployed for an alternating biopanning using a sunflower trypsin inhibitor1-based phage display (SFTI8Ph) library. Stability, binding properties and affinity of novel candidates were assessed in vitro using radio-HPLC, binding experiments and surface plasmon resonance assay (SPR), respectively. Subsequently, the affinity of the peptide was verified in situ by using peptide histochemistry, in vitro using flow cytometry, and in vivo by positron emissions tomography (PET/CT).Results: We identified a novel ITGαvβ6 binding peptide (SFITGv6) containing the amino acid sequence FRGDLMQL. SFITGv6 provides stability over a period of 24 hours and demonstrates high affinity (KD = 14.8 nmol/L) for ITGαvβ6 In HNO97 cells, a maximal uptake and internalization of up to 37.3% and 37.5%, respectively, was measured. Small-animal PET imaging and biodistribution studies of HNO97 xenografted Balb/c nu/nu mice showed tumor-specific accumulation of 68Ga- and 177Lu-labeled DOTA-SFITGv6, respectively, 30 to 60 minutes after injection. Moreover, peptide histochemistry revealed a strong and homogenous binding of biotin-labeled SFITGv6 to HNSCC tumors and breast- and lung cancer-derived brain metastases. Finally, first PET/CT scans of HNSCC and NSCLC patients displayed SFITGv6 accumulation specifically in tumors, but not in inflammatory lesions.Conclusions: Thus, SFITGv6 represents a novel powerful tracer for imaging and possibly for endoradiotherapy of ITGαvβ6-positive carcinoma. Clin Cancer Res; 23(15); 4170-80. ©2017 AACR.

Highlights of This Issue

Defects in homologous recombination repair genes (e.g., BRCA1 and BRCA2 mutations) can sensitize tumors to poly(ADP-ribose) polymerase (PARP) inhibition through synthetic lethality. This phase I–II study was the first to evaluate single-agent oral rucaparib, a potent PARP inhibitor, at multiple doses in patients with advanced solid tumors (Part 1; phase I), establish the recommended phase II dose (RP2D) of 600 mg twice daily, and evaluate the RP2D in patients with platinum-sensitive, high-grade ovarian carcinoma (HGOC) associated with a germline BRCA1/2 mutation (Part 2A; phase II). Rucaparib was tolerable and had robust antitumor activity in patients with platinum-sensitive germline BRCA1/2-mutated HGOC.Germinal center B-cell (GCB) has a better prognosis than non-GCB diffuse large B-cell lymphoma (DLBCL) to upfront therapy. Cell-of-origin (COO) determination using gene expression profiling (GEP) is more sensitive, reproducible, and robust than immunohistochemistry. Czuczman and colleagues designed a randomized study using COO-based subtyping to elucidate differences in activity of lenalidomide over salvage monotherapy in relapsed/refractory DLBCL. Lenalidomide elicited higher ORR and longer PFS in non-GCB patients (most pronounced in ABC-designated patients). These results provide an additional rationale for the ongoing phase 3 trial, ROBUST (NCT02285062), of lenalidomide plus immunochemotherapy versus immunochemotherapy alone in patients with treatment-naive GEP-selected ABC-subtype DLBCL.The improvement of imaging and therapeutic targeting of tumors requires the identification of novel target-specific tracers. To address HNSCC tumors, Altmann and colleagues performed alternating biopanning on membrane proteome of HNO97 tumor cells fractionated by the ProteomeLab PF2D system and corresponding HNO97 cells using a sunflower trypsin inhibitor1-based phage display (SFTI8Ph) library. A novel ITGαvβ6-binding peptide (SFITGv6) was identified providing high stability and demonstrating affinity for ITGαvβ6-positive HNSCC and other carcinomas. SFITGv6 accumulated in tumor tissues but not in normal tissues or inflammatory lesions, and thus represents a promising tracer for imaging and endoradiotherapy.Deubiquitylating (DUB) enzymes have emerged as potential therapeutic targets. Das and colleagues examined the functional significance of DUB enzyme USP1 in multiple myeloma (MM) using an irreversible USP1 inhibitor SJB3-019A. USP1 is highly expressed in patient multiple myeloma (MM) cells. USP1 blockade with SJB3-019A decreases the viability of MM cells, inhibits DNA repair and USP1/ID/Notch/Sox pathways, and overcomes bortezomib-resistance. SJB induced generation of more mature and differentiated plasma cells. Combination of SJB with bortezomib, ACY1215, lenalidomide, or pomalidomide triggers synergistic anti-MM activity. These preclinical data provide basis for clinical evaluation of USP1 inhibitors in MM to overcome proteasome inhibitor resistance.

Proteome profiling of the compatible interaction between wheat and stripe rust

Over the last decade, comparative molecular profiling studies between compatible and incompatible plant-pathogen interactions have shown that susceptible response of the host to a pathogen requires factors that promote disease development. In this study, we examined proteome profiles during a compatible interaction between wheat and stripe rust. A 2D-LC system (ProteomeLab PF2D) was used for protein separation and to compare the proteome from infected and control samples. More than 700 protein peaks at each time point were compared between pathogen- and mock-inoculated samples. Selected proteins, with significant differences in abundance were identified by nanoLC-ESI- MS/MS and generated spectra were searched against the wheat protein databases from UniProt, and NCBI and the Puccinia database from The Broad Institute. In total, the identified proteins comprised of 62 % wheat and 38 % Pst proteins. All identified proteins were searched by bioinformatics-based algorithms to detect their subcellular localization and signal peptide motifs which have the potential to catch the candidate effector proteins. The wheat proteins were classified based on their function. Although a compatible interaction, many wheat proteins, such as antioxidants, PRs and cold-responsive proteins, are implicated in defense and stress tolerance. On the pathogen side, 64 proteins were identified, and included some important pathogenicity proteins that can play role in pathogen virulence and suppress the host defense. In addition, we discovered that nine proteins have a signal sequence and three of the hypothetical fungal proteins, PGTG_11681T0, PGTG_07231T0 and CBH50687.1, have been tentatively identified as candidate effectors.

A Novel Proteomic Analysis of the Modifications Induced by High Hydrostatic Pressure on Hazelnut Water-Soluble Proteins.

Food allergies to hazelnut represent an important health problem in industrialized countries because of their high prevalence and severity. Food allergenicity can be changed by several processing procedures since food proteins may undergo modifications which could alter immunoreactivity. High-hydrostatic pressure (HHP) is an emerging processing technology used to develop novel and high-quality foods. The effect of HHP on allergenicity is currently being investigated through changes in protein structure. Our aim is to evaluate the effect of HHP on the protein profile of hazelnut immunoreactive extracts by comparative proteomic analysis with ProteomeLab PF-2D liquid chromatography and mass spectrometry. This protein fractionation method resolves proteins by isoelectric point and hydrophobicity in the first and second dimension, respectively. Second dimension chromatogram analyses show that some protein peaks present in unpressurized hazelnut must be unsolubilized and are not present in HHP-treated hazelnut extracts. Our results show that HHP treatment at low temperature induced marked changes on hazelnut water-soluble protein profile.

Proteomic identification of angiomotin by ProteomeLab PF-2D and correlation with clinical outcome in human clear cell renal cell carcinoma

Identification of new therapeutic and prognostic biomarkers for clear cell renal cell carcinoma (ccRCC) is urgently required since most patients are in advanced stages of ccRCC at initial diagnosis and the recurrence rate is high. Differentially expressed proteins between the ccRCC cell line RLC-310 and the normal renal cell line HK-2 were identified by a comparative proteomic approach based on a protein fractionation two-dimensional (PF-2D) liquid-phase fractionation system and capillary liquid chromatography electrospray ionization mass spectrometry/mass spectrometry (LC-ESI-MS/MS). Differentially expressed proteins (n=196) were identified. Of the 13 differentially expressed proteins newly discovered in ccRCC, angiomotin (Amot) was the focus of this study since its role in ccRCC progression has been obscure. The overexpression of Amot in ccRCC tissues was confirmed by comparing Amot expression in 18 primary ccRCC tissues and adjacent normal renal tissues (ANRT) using western blot analysis. Quantitative RT-PCR using 127 ccRCC tissues revealed that high levels of Amot transcripts were associated with poor differentiation, venous invasion and decreased survival (p<0.0001, <0.05 and <0.05). Multivariate analysis indicated that Amot transcript was an independent prognostic factor for the survival of ccRCC patients (p<0.05). These data suggest that Amot may serve as a novel prognostic factor of ccRCC.

Serum Proteomic Signature of Human Chagasic Patients for the Identification of Novel Potential Protein Biomarkers of Disease

Chagas disease is initiated upon infection by Trypanosoma cruzi. Among the health consequences is a decline in heart function, and the pathophysiological mechanisms underlying this manifestation are not well understood. To explore the possible mechanisms, we employed IgY LC10 affinity chromatography in conjunction with ProteomeLab PF2D and two-dimensional gel electrophoresis to resolve the proteome signature of high and low abundance serum proteins in chagasic patients. MALDI-TOF MS/MS analysis yielded 80 and 14 differentially expressed proteins associated with cardiomyopathy of chagasic and other etiologies, respectively. The extent of oxidative stress-induced carbonyl modifications of the differentially expressed proteins (n = 26) was increased and coupled with a depression of antioxidant proteins. Functional annotation of the top networks developed by ingenuity pathway analysis of proteome database identified dysregulation of inflammation/acute phase response signaling and lipid metabolism relevant to production of prostaglandins and arachidonic acid in chagasic patients. Overlay of the major networks identified prothrombin and plasminogen at a nodal position with connectivity to proteome signature indicative of heart disease (i.e., thrombosis, angiogenesis, vasodilatation of blood vessels or the aorta, and increased permeability of blood vessel and endothelial tubes), and inflammatory responses (e.g., platelet aggregation, complement activation, and phagocyte activation and migration). The detection of cardiac proteins (myosin light chain 2 and myosin heavy chain 11) and increased levels of vinculin and plasminogen provided a comprehensive set of biomarkers of cardiac muscle injury and development of clinical Chagas disease in human patients. These results provide an impetus for biomarker validation in large cohorts of clinically characterized chagasic patients.

Preliminary analysis of differentially expressed proteins of clear-cell renal cell carcinoma by comparative proteomic technologies

To explore the different expression of proteins between human clear-cell renal cell carcinoma (ccRCC) cell line RLC-310 and normal renal proximal tubule epithelial cell line HK-2, and to search new differentially expressed proteins of RCC. RLC-310 and HK-2 cells were cultured in vitro. The total proteins were separated by ProteomeLab PF 2D protein fractionation system and the differential expression protein fractions of the two cell lines were analyzed and identified by capillary liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS/MS). RT-PCR and Western blot were used to confirm the representative differential expression at mRNA and protein levels. One hundred and ninty-six differentially expressed proteins were identified. These differentially expressed proteins involved in many aspects, including cell proliferation and anti-apoptosis, energy metabolism, mitochondria reduction and oxidation, oxidative stress and resistance, cell signaling, invasion and adhesion, cytoskeleton and motion, neovascularization, etc. Except for previously reported RCC associated proteins: annexin A2, fatty acid-binding protein, vimentin, fibronectin, and so on, Septin-9 was firstly found highly expressed in RLC-310 cells when compared with that in the HK-2 cells. Moreover, the overexpression of Septin-9 was confirmed by RT-PCR and Western blot analysis at both mRNA and protein levels (P < 0.05). The human ccRCC cell line RLC-310 cells display differential protein profiles compared with those of the normal renal cell line HK-2 cells. The identified differential expression proteins are involved in many aspects of RCC development. It is worth further study and elucidate the molecular mechanisms of RCC. The representative differential protein Septin-9 deserves further study its role in the angiogenesis of ccRCC.

Immunoproteomics Identification of Major IgE and IgG4 Reactive Schistosoma japonicum Adult Worm Antigens Using Chronically Infected Human Plasma.

Immunoepidemiological studies from endemic areas have revealed age-dependent resistance correlation with increased level of IgE and decreased level of IgG4 antibodies in responses to schistosomes’ soluble worm antigen. However, there have been limited studies on analyses of major antigens that provoke IgE and IgG4 immune response during chronic stage of schistosomiasis. In this study, for the first time, immunoproteomics approach has been applied to identify S. japonicum worm antigens in liquid fractions that are recognized by IgE and IgG4 antibody using plasma from chronically infected population. ProteomeLabPF 2D fractionated 1-D and 2-D fractions of SWA antigens were screened using pooled high IgE/IgG4 reactive plasma samples by dot-blot technique. In 1-D fractions, IgE isotype was detected by fewer antigenic fractions (43.2%). The most recognized isotype was IgG3 (79.5%) followed by IgG1 (75.0%) and IgG4 (61.4%). Liquid chromatography MS/MS protein sequencing of reactive 2-D fractions revealed 18 proteins that were identified, characterized and gene ontology categories determined. 2-D fractions containing proteins such as zinc finger, RanBP2-type, domain-containing protein were strongly recognized by IgE and moderately by IgG4 whereas fractions containing proteins such as ubiquitin-conjugating enzyme and cytosolic II 5'-nucleotidase strongly recognizing by IgG subclasses (IgG1, IgG3 and IgG4) but not IgE. By this study, a simple and reproducible proteomic method has been established to identify major immunoreactive S. japonicum antigens. It is anticipated that this will stimulate further research on the immunogenicity and protective potential of proteins identified as well as discovery of novel compounds that have therapeutic importance.

Proteomic analysis of the Arabidopsis thaliana-Botrytis cinerea interaction using two-dimensional liquid chromatography

A two-dimensional liquid chromatography (2D LC) system, ProteomeLab PF 2D, was employed to study the defence proteome of Arabidopsis following infection with the necrotrophic fungal pathogen, Botrytis cinerea . This system demonstrated differential protein expression in control and treated samples in some fractions. However, the amount of proteins in the fractions and the level to which they were changing could not be established. Among the proteins identified in the fractions that displayed an increase in absorbance was catalase 3 and glutathione S-transferases, demonstrating the importance of an antioxidant system in defence against B. cinerea. Most of the proteins were identified in fractions that displayed reduction in absorbance. Functional categorisation of the identified proteins demonstrated the overrepresentation of photosynthetic pathway, a phenomenon also observed in other host and nonhost pathogen interactions. Proteomelab PF 2D did not identify as many proteins as expected possibly due to the masking effect of ribulose-1,5-bisphosphate carboxylase oxygenase (RuBisCO) as this protein was identified in almost all fractions including those having an increase in absorbance. Depletion of this protein from crude plant protein extracts is likely to improve protein identification by mass spectrometry, especially for the low abundant proteins. A number of proteins were identified in each fraction and it was difficult to discern which of the proteins was responsible for the differential increase or reduction in absorbance. Keywords: Botrytis cinerea, Arabidopsis thaliana, necrotroph, proteome, ProteomeLab PF2D.

A preliminary study on screening for Porphyromonas gingivalis outer membrane protein antigen with two-dimensional liquid phase fractionation and matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry

To screen a variety of Porphyromonas gingivalis (Pg) common outer membrane proteins with two-dimensional liquid phase fractionation (PF2D) and matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF-MS) and provide candidate target antigen for the design of vaccines with cross protection against a variety of Pg. The outer membrane proteins of Pg301, PgATCC33277 and PgW83 were extracted through ultracentrifugation, and then they were separated by ProteomeLab PF2D protein fractionation system. After separation, the outer membrane proteins were obtained through comparison, and the primary structure of the proteins was identified by MALDI-TOF/TOF-MS and database. Ninety-nine protein samples out of 3 strains of Pg were obtained after the high performance chromato focusing (HPCF) separation process. B7 fractions of 3 strains of Pg were separated by the reversed-phase high performance liquid chromatography (RP-HPCL) separation process. After comparison of peak and retention time of chromatogram, the 8 common protein peaks of 3 strains of Pg were confirmed. The protein samples were identified by MALDI-TOF/TOF-MS, and one of them was known protein arg-gingipain A. PF2D protein fractionation system is of good reproducibility and high resolution. A combination of PF2D and MALDI-TOF/TOF-MS can be used to identify the common outer membrane proteins of Pg.

Proteomic analysis of wheat embryos with 2-DE and liquid-phase chromatography (ProteomeLab PF-2D) — A wider perspective of the proteome

Cereal embryos are a model system to study desiccation tolerance due to their ability to survive extreme water loss during late embryogenesis. To identify proteins accumulating in mature embryos which can be used as potential markers for dehydration tolerance, we compared the embryo proteome from two durum wheat genotypes (Triticum durum Desf.), Mahmoudi (salt and drought sensitive) and Om Rabia3 (salt and drought tolerant). Total protein extracts from wheat embryos were analyzed by using conventional 2-DE and ProteomeLab PF-2D. Analysis using different pH ranges showed that a larger number of fractions were solved by LC, than by conventional 2-DE at extreme technical pHs (pH 4.0-5.0 and pH 6.5-8.0). In contrast, at intermediate pHs (pH 5.0-6.5), resolution was better in 2-DE gels. The two techniques were used in parallel to analyze total protein extracts from embryos of the two wheat varieties. Several proteins belonging to the seed storage family, LEA-type/heat shock proteins, enzyme metabolism and radical scavengers were identified by analysis of trypsin digested peptides via mass spectrometry. These proteins accumulate in different amounts in embryos of tolerant and sensitive wheat varieties. The differences in expression pattern were further validated by enzyme activity, western blotting analysis and correlated with their corresponding mRNA expression by RT-PCR analyses for the corresponding protein. We suggest that the differential expression pattern could be used as a basis for a biochemical screen of tolerance/sensitivity to drought and salt stress in wheat embryos and germplasm.

Abstract A32: Proteomic signatures of deoxycholic acid and ursodeoxycholic acid treated human colon cancer cells

Abstract A32 Dietary factors play an important role in the pathogenesis of colorectal cancer (CRC), due in part to the influence of the bile acids. While the secondary bile acid, deoxycholic acid (DCA), has been associated with tumor promotion, one tertiary bile acid in particular, ursodeoxycholic acid (UDCA), has been demonstrated to exert chemopreventive properties in the colon. Although its efficacy against high-grade adenomas has already been demonstrated in phase III clinical trials, mechanisms by which UDCA suppresses colon tumorigenesis remain unclear. In order to gain further insight into the tumor modifying properties of the bile acids, we compared the effects of DCA and UDCA on human colon tumor cells (CaCo-2) using proteomic analyses. In the following study, CaCo-2 cells grown at 70% confluence were treated with either 10 µM deoxycholic acid (DCA) or 1 mM ursodeoxycholic acid (UDCA) for 72 hours. For the proteomic analysis, protein was first fractionated by PF2D isochromatofocusing on a Beckman Coulter ProteomeLab PF2D platform. Fractions corresponding to a linear gradient between pH 8.0 and 4.0 were collected and processed through an automated autoloader for further separation by HPRP-PF2D reverse phase chromatography. Two dimensional protein expression maps displaying isoelectric point (pI) versus hydrophobicity were generated by the ProteoView/DeltaVue software package. Pairwise analysis of these complex protein chromatograms demonstrated common peak profiles (signatures) as well as unique peaks (fingerprints) characteristic of UDCA or DCA treatment. One of these protein peaks that exhibited a differential expression pattern between the two drug treatments was identified by MALDI and tandem mass spectrometry sequencing as heat-shock protein 60 (Hsp60). Furthermore, this protein was found to be less oxidized upon treatment with UDCA, suggesting the attenuation of oxidative stress present in cancer cells. Further analyses on the role of Hsp60 as well as resultant diminished oxidative stress will enable the discovery of potential mechanisms by which DCA and UDCA control cellular pathways. Citation Information: Cancer Prev Res 2008;1(7 Suppl):A32.

A comparison of 2‐D chromatography separations using UV and 18O quantification of proteins in similar proteomes

Proteins present in two mitochondria preparations were separated by 2-D chromatography using the ProteomeLab PF-2D Protein Fractional System, protein fractionation in two dimensions (PF-2D). The proteins in each first-dimension fraction were determined by trypsinization and LC-MS/MS. Chromatography peaks were quantified by UV detection using the "Mapping Tools" software (Beckman). The proteins present in UV detected peaks were trypsinized and identified by automated MS/MS sequencing. Relative amounts of the proteins present in the equivalent peak for each sample were assessed by comparison of the intensities of the constituent peptides and a predicted PF-2D value was calculated from the total ion count (TIC) for each peptide. Relative quantification for (18)O labeled peptides was performed using the ZoomQuant (v1.43b) software [1, 2]. We found that the chromatography peaks detected by UV generally contained several proteins. Using (18)O labeling we determined that in each peak the ratios of the constituent proteins were different. When these ratios were normalized using the TIC to account for abundance, the resulting ratio corresponded to that determined by UV. The predicted value for the PF-2D score corresponded to the observed value for each peak irrespective of the number or proteins detected.

Utilizing endoscopic technology to reveal real‐time proteomic alterations in response to chemoprevention

Cancer chemoprevention approaches use either pharmacological or dietary agents to impede, arrest or reverse the carcinogenic process. Although several agents have shown effectiveness against colon cancer, present intervention strategies provide only partial reduction. In this study, we utilized high-resolution endoscopy to obtain colon tumor biopsy specimens from Apc mutant mice before and after 2-wk sulindac intervention. To acquire information beyond genomics, proteome analysis using the ProteomeLab PF2D platform was implemented to generate 2-D protein expression maps from biopsies. Chromatograms produced common signature profiles between sulindac and nonsulindac treated samples, and contrasting profiles termed "fingerprints". We selected a double peak that appeared in tumor biopsies from sulindac-treated mice. Further analyses using MS sequencing identified this protein as histone H2B. The location of H2B in the 1(st) dimension strongly suggested PTM, consistent with identification of two oxidized methionines. While further studies on sulindac proteomic fingerprints are underway, this study demonstrates the feasibility and advantages of "real-time" proteomic analysis for obtaining information on biomarker discovery and drug activity that would not be revealed by a genetic assay. This approach should be broadly applicable for assessing lesion responsiveness in a wide range of translational and human clinical studies.

Relative quantitation of proteins fractionated by the ProteomeLab™ PF 2D system using isobaric tags for relative and absolute quantitation (iTRAQ)

We describe an optimised protocol for application of isobaric tags for relative and absolute quantitation (iTRAQ) and tandem mass spectrometry to obtain relative quantitative data from peptides derived from tryptic digestions of proteins fractionated by using the 2D liquid-phase ProteomeLab PF 2D technique. This methodology is suitable for the quantitation of proteins from a pool of co-eluting proteins which are often difficult to identify for the purpose of candidate protein selection for biologically relevant qualitative/quantitative changes under experimental conditions or in disease states. iTRAQ quantitation also facilitates the possibility of result to result comparison using other methodologies such as UV protein quantitation via the ProteomeLab PF 2D technique. The optimised protocol outlined here allows relative quantitation by MALDI-TOF/TOF mass spectrometry with high sensitivity and without the need to perform 2D HPLC separation of labelled peptides. The overall outcome is the simplification in the data complexity and the ease of use of the labelling protocol.

Identification and Quantification of Basic and Acidic Proteins Using Solution-Based Two-Dimensional Protein Fractionation and Label-Free or 18O-Labeling Mass Spectrometry

Reduction in sample complexity enables more thorough proteomic analysis using mass spectrometry (MS). A solution-based two-dimensional (2D) protein fractionation system, ProteomeLab PF 2D, has recently become available for sample fractionation and complexity reduction. PF 2D resolves proteins by isoelectric point (pI) and hydrophobicity in the first and second dimensions, respectively. It offers distinctive advantages over 2D gel electrophoresis with respects to automation of the fractionation processes and characterization of proteins having extreme pIs. Besides fractionation, PF 2D is equipped with built-in UV detectors intended for relative quantification of proteins in contrasting samples using its software tools. In this study, we utilized PF 2D for the identification of basic and acidic proteins in mammalian cells, which are generally under-characterized. In addition, mass spectrometric methods (label-free and 18O-labeling) were employed to complement protein quantification based on UV absorbance. Our studies indicate that the selection of chromatographic fractions could impact protein identification and that the UV-based quantification for contrasting complex proteomes is constrained by coelution or partial coelution of proteins. In contrast, the quantification post PF 2D chromatography based on label-free or 18O-labeling mass spectrometry provides an alternative platform for basic/acidic protein identification and quantification. With the use of HCT116 colon carcinoma cells, a total of 305 basic and 183 acidic proteins was identified. Quantitative proteomics revealed that 17 of these proteins were differentially expressed in HCT116 p53-/- cells.

An Immuno-PF2D-MS/MS Proteomic Approach for Bacterial Antigenic Characterization: To Bacillus and Beyond

We are confronted daily to unknown microorganisms that have yet to be characterized, detected, and/or analyzed. We propose, in this study, a multidimensional strategy using polyclonal antibodies, consisting of a novel proteomic tool, the ProteomeLab PF2D, coupled to immunological techniques and mass spectrometry (i-PF2D-MS/MS). To evaluate this strategy, we have applied it to Bacillus subtilis, considered here as our unknown bacterial model.

Improved protocol for chromatofocusing on the ProteomeLab PF2D

Beckman-Coulter has recently introduced the ProteomeLab PF2D for 2-D liquid separation of protein samples. The system features separation in the first dimension by chromatofocusing, followed by RP chromatography in the second dimension, allowing the analysis of complex proteomics samples. When used by the standard protocol, reproducibility and column life times are limited, making the use of the instrument very costly. We here present an improved protocol for chromatofocusing, which enhances column life by at least fivefold.

Proteomic analysis of enamel matrix using a two‐dimensional protein fractionation system

Our objectives in this study were to perform separate proteomic analyses of porcine soft and hard enamel matrices, using the ProteomeLab PF-2D System, to compare the contents of the hard and soft enamel and to identify matrix constituents that are absent from the early maturation stage. Developing first permanent molars were dissected from 6-month-old pigs. Both immature and mature enamel samples were obtained by scraping the secretory-stage (soft) and maturation-stage (hard) enamel, respectively. Enamel matrix samples were sequentially extracted and fractionated with 50 mM phosphate buffer (pH 7.4) and then with 50 mM carbonate buffer (pH 10.8). The neutral enamel extract was separated into four fractions by successive ammonium sulfate precipitations. The alkaline enamel extract was separated into four fractions by ion-exchange chromatography. These eight extracts from both the soft and hard enamel were injected for chromatofocusing. Soft enamel fractions containing constituents absent from the hard enamel were further separated by reverse-phase high-performance liquid chromatography. The major soft enamel constituents absent from the hard enamel were acidic glycoproteins, corresponding to the 32-kDa enamelin, and the 29-, 27-, 15-, 13-, 8- and 6-kDa C-terminal fragments of ameloblastin. Loss of these glycoproteins is associated with a post-transition increase in enamel mineralization.

A 2-D liquid-phase chromatography for proteomic analysis in plant tissues☆

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.