Proteomic analysis in human breast cancer: identification of a characteristic protein expression profile of malignant breast epithelium

Gene expression analysis has become a promising tool in predicting the clinical course of malignant disease and the response to antineoplastic therapy. Surprisingly, only little is known about the protein expression pattern of human tumors. Recent advances in proteomic analysis allow proteins of interest to be identified by their expression and/or modification pattern in 2-DE rather than using the traditional approach of translating gene expression data. To identify a proteomic pattern that is characteristic for malignant breast epithelium, we performed differential 2-DE analysis in sets of microdissected malignant breast epithelia and corresponding adjacent normal breast epithelia from five patients with invasive breast carcinoma. Thirty-two protein spots were found to be selectively regulated in malignant epithelium, and were subjected to MALDI-TOF and/or immunoblotting for protein identification. Thirteen of the identified proteins had previously not been associated with breast cancer. The validity of these findings was confirmed by literature review and immunohistochemistry for identified proteins in an independent cohort of 50 breast cancer specimens. We here describe, for the first time, a proteomic analysis of matched normal and malignant epithelia from invasive breast carcinomas. This strategy leads to a better understanding of oncogenesis at an operational level and helps to characterize the malignant phenotype of individual tumors, and thereby to identify novel targets for antineoplastic therapy.     

Wheat proteomics: relationship between fine chromosome deletion and protein expression

To explore the relationship between fine chromosome deletion and protein expression in common wheat, the changes in protein composition of wheat seed proteome were investigated by using chromosome 1B. A momosomic alien chromosome addition line of common wheat was used to produce the fine deletion lines. Endosperm and embryo proteins were separated by two-dimensional gel electrophoresis (2-DE) and visualized by staining with Commassie Brilliant Blue, and gel images were analyzed with a computer assisted image analyzer. For the first time, fine gene locations of a few endosperm and embryo proteins were identified on the chromosome 1B. These proteins with their specific gene location on the chromosome can be used as protein markers in breeding programs for quality of wheat proteins. To identify wheat seed proteins and to understand their expression in relation to chromosome deletion, the feasibility of a new analytical approach based on isotope coded affinity tag labeling (ICAT) of peptides in tryptic digests followed by electrospray ionization mass spectrometry has been described. Simplification of the complex tryptic digest prior to mass spectral analysis was performed by treating the samples with light and heavy ICAT labeling reagents. A clear separation of peptide fragment containing the light and heavy reagents was achieved in mass spectral analysis. Out of the 14 peptides detected by mass fragment analysis of the euploid, four were down-regulated, nine up-regulated and one did not show any change due to the terminal deletion of chromosome 1B. Selected peptide fragments were subjected to tandem mass spectrometry analysis for sequence information and the resulting sequence information was submitted to databases for protein identification. Of the five proteins submitted, four were identified as alpha-amylase inhibitor, alpha-amylase/subtilisin inhibitor precursor, proteasome subunit alpha-type 7 and 1,4 alpha-glucan-D-maltohydrolase. With this approach it is possible to identify wheat seed proteins and to understand their expression, which have been reported to be difficult by 2-DE due to cosynthesis of proteins by genes from three genomes, A, B and D.     

A comparative evaluation of software for the analysis of liquid chromatography-tandem mass spectrometry data from isotope coded affinity tag experiments

The options available for processing quantitative data from isotope coded affinity tag (ICAT) experiments have mostly been confined to software specific to the instrument of acquisition. However, recent developments with data format conversion have subsequently increased such processing opportunities. In the present study, data sets from ICAT experiments, analysed with liquid chromatography/tandem mass spectrometry (MS/MS), using an Applied Biosystems QSTAR Pulsar quadrupole-TOF mass spectrometer, were processed in triplicate using separate mass spectrometry software packages. The programs Pro ICAT, Spectrum Mill and SEQUEST with XPRESS were employed. Attention was paid towards the extent of common identification and agreement of quantitative results, with additional interest in the flexibility and productivity of these programs. The comparisons were made with data from the analysis of a specifically prepared test mixture, nine proteins at a range of relative concentration ratios from 0.1 to 10 (light to heavy labelled forms), as a known control, and data selected from an ICAT study involving the measurement of cytokine induced protein expression in human lymphoblasts, as an applied example. Dissimilarities were detected in peptide identification that reflected how the associated scoring parameters favoured information from the MS/MS data sets. Accordingly, there were differences in the numbers of peptides and protein identifications, although from these it was apparent that both confirmatory and complementary information was present. In the quantitative results from the three programs, no statistically significant differences were observed.     

Differentially isotope-coded N-terminal protein sulphonation: combining protein identification and quantification

Most proteomic labelling technologies intend to improve protein quantification and/or facilitate (de novo) peptide sequencing. We present here a novel stable-isotope labelling method to simultaneously identify and quantify protein components in complex mixtures by specifically derivatizing the N-terminus of proteins with 4-sulphophenyl isothiocyanate (SPITC). Our approach combines protein identification with quantification through differential isotope-coded labelling at the protein N-terminus prior to digestion. The isotope spacing of 6 Da (unlabelled vs. six-fold 13C-labelled tag) between derivatized peptide pairs enables the detection on different MS platforms (MALDI and ESI). Optimisation of the reaction conditions using SPITC was performed on three model proteins. Improved detection of the N-terminally derivatized peptide compared to the native analogue was observed in negative-ion MALDI-MS. Simpler fragmentation patterns compared to native peptides facilitated protein identification. The 13C-labelled SPITC resulted in convenient peptide pair spacing without isotopic overlap and hence facilitated relative quantification by MALDI-TOF/TOF and LC-ESI-MS/MS. The combination of facilitated identification and quantification achieved by differentially isotope-coded N-terminal protein tagging with light/heavy SPITC represents, to our knowledge, a new approach to quantitative proteomics.     

A quantitative proteomic analysis of growth factor-induced compositional changes in lipid rafts of human smooth muscle cells

Signals that promote proliferation and migration of smooth muscle cells (SMC) have been implicated in pathologic growth of hollow organs. Members of the platelet-derived growth factor (PDGF) family, potent mitogens and motility factors for SMC, have been shown to signal through cholesterol-enriched lipid rafts. We recently demonstrated that PDGF-stimulated DNA synthesis in urinary tract SMC was dependent on the integrity of lipid rafts. Despite its known ability to rapidly alter discrete proteins within rafts, the effect of PDGF on overall raft protein composition is unknown. In this study, we employed isotope coded affinity tag (ICAT) analysis to evaluate PDGF-induced protein changes in lipid rafts of primary culture human SMC. Following acute (i.e., 15 min) exposure of SMC to PDGF, 23 proteins increased in rafts >20%. In contrast, raft localization of only three proteins increased after 12 h of PDGF treatment. Among the proteins that increased at 15 min were the glycophosphatidylinositol-anchored proteins Thy-1, 5'-nucleotidase, and CD55, the cytoskeletal proteins actin, actinin, tropomyosin-3 and -4, and the endocytosis-related proteins clathrin and beta-adaptin. In addition, eight Rho family members were localized to rafts by ICAT analysis. Collectively, these observations suggest a role for lipid rafts in regulation of PDGF-stimulated changes in the cytoskeleton.     

Relative and absolute quantitative expression profiling of cytochromes P450 using isotope-coded affinity tags

The development of a novel method for absolute quantification of proteins based on isotope-coded affinity tagging using ICAT reagents is described. The method exploits synthetic peptide standards to determine protein content at the femtomole level in biological samples. The approach is generally applicable to any subset of proteins, but is particularly appropriate for quantitative analysis of multiple, closely related isoforms, and for hydrophobic proteins that are poorly represented in 2-D gels. Relative and absolute quantification techniques are applied to an important group of microsomal metabolic enzymes, the cytochromes P450 (P450), which are critical in determining the disposition, safety and efficacy of drugs in man. Measurement of the P450 induction profile in response to chemicals is a fundamental aspect of drug safety evaluation and is currently achieved by low-throughput methods employing poorly discriminatory antibodies or substrates. Tagging technology is shown to supersede conventional methods for P450 profiling in terms of discriminatory power and throughput, exemplified by the simultaneous detection of distinct induction profiles for cyp2c subfamily members in response to phenobarbitone: cyp2c29 expression, but not cyp2c40 or cyp2c50, was induced threefold by treatment. This technology should abbreviate the drug development pathway, and provide a widely applicable, rapid means of quantifying proteins.     

Proteome analysis of secreted proteins during osteoclast differentiation using two different methods: two-dimensional electrophoresis and isotope-coded affinity tags analysis with two-dimensional chromatography

Bone is maintained by two cell types, bone-forming osteoblasts and bone-resorbing osteoclasts. Osteoblasts express two factors, osteoprotegerin and receptor activator of NF-kappaB ligand (RANKL), inhibiting and promoting osteoclast differentiation, respectively. In contrast, modulators of bone resorption expressed by osteoclasts have not been so well studied enough. In the present study, we demonstrate proteome analysis of secreted proteins during osteoclast differentiation to elucidate the molecular mechanism of bone resorption and bone remodeling. To achieve this objective, we chose RAW264.7 cells with RANKL as a homogeneous osteoclast differentiation model and used two methods, two-dimensional gel electrophoresis (2-DE) and isotope-coded affinity tags (ICAT) analysis with two-dimensional liquid chromatography. We found 23 spots in 2-DE and 19 proteins in ICAT analysis which were expressed differently during osteoclast differentiation. These two methods gave us closely related but different information about proteins, suggesting they are complementary or at least supplementary methods at present. Cathepsins, osteopontin, legumain, macrophage inflammatory protein-1alpha, and other proteins were observed as up- or down-regulated proteins and are discussed in the context of osteoclast differentiation and bone resorption. In addition to confirming previous observations, this study indicates novel proteins related to osteoclast differentiation which are potential therapeutic targets for the treatment of bone diseases, such as osteoporosis.     

Comprehensive quantitative proteome analysis of 20S proteasome subtypes from rat liver by isotope coded affinity tag and 2-D gel-based approaches

Quantitative protein profiling is an essential part of proteomics and requires technologies that accurately, reproducibly, and comprehensively identify and quantify proteins. Over the past years, many quantitative proteomic methods have been developed. Here, 20S proteasome subtypes isolated from rat were compared by four approaches based on the combination of isotope-coded affinity tag (ICAT), 2-DE, LC and ESI and MALDI MS: (i) 2-DE, (ii) ICAT/2-DE MALDI-MS, (iii) ICAT/LC-ESI-MS, (iv) ICAT/LC-MALDI-MS. A definite qualitative advantage of 2-DE gels was the separation of all known protein species, the identification of cysteine sulfoxide of alpha-4 (RC6-IS) and N-terminal acetylation of several subunits. Furthermore, quantitative differences between the standard subunits beta-2, and beta-5 and their immunosubunits were only detected by 2-DE image analysis revealing a higher replacement of standard- by immuno-beta-subunits in subtype IV. It was obvious that for relative quantification only protein spot and mass peaks with a certain level of intensity displayed acceptable values of SD. However, ICAT in conjunction with LC/MALDI-MS was the most accurate method for quantification. The experimental data of this investigation are accessible via http://www.mpiib-berlin.mpg.de/2D-PAGE/.     

Mass spectrometry-based proteomic analysis of the epitope-tag affinity purified protein complexes in eukaryotes

In recent years, MS has been widely used to study protein complex in eukaryotes. The identification of interacting proteins of a particular target protein may help defining protein-protein interaction and proteins of unknown functions. To isolate protein complexes, high-speed ultracentrifugation, sucrose density-gradient centrifugation, and coimmunoprecipitation have been widely used. However, the probability of getting nonspecific binding is comparatively high. Alternatively, by use of one- or two-step (tandem affinity purification) epitope-tag affinity purification, protein complexes can be isolated by affinity or immunoaffinity columns. These epitope-tags include protein A, hexahistidine (His), c-Myc, hemaglutinin (HA), calmodulin-binding protein, FLAG, maltose-binding protein, Strep, etc. The isolated protein complex can then be subjected to protease (i.e., trypsin) digestion followed by an MS analysis for protein identification. An example, the epitope-tag purification of the Arabidopsis cytosolic ribosomes, is addressed in this article to show the success of the application. Several representative protein complexes in eukaryotes been isolated and characterized by use of this approach are listed. In this review, the comparison among different tag systems, validation of interacting relationship, and choices of MS analysis method are addressed. The successful rate, advantages, limitations, and challenges of the epitope-tag purification are also discussed.     

Mass profiling-directed isolation and identification of a stage-specific serologic protein biomarker of advanced prostate cancer

Carcinoma of the prostate (CaP) is the second leading cause of cancer-related mortality among American men. While high cure rates are associated with localized CaP, no cure exists for advanced recurrent disease. At present there are no known serologic biomarkers specific to this stage of the disease. Several groups have used mass spectrometry (MS) based mass profiling (MP) combined with multivariate analysis to identify diagnostically predictive protein peaks for CaP in serum and tissues. Nevertheless, an appreciable level of skepticism exists for MP attributed primarily to a lack of definitive protein characterization. To address this problem, we have applied an approach that combines MP with a whole-protein based top-down separation strategy for the identification of a stage-specific marker in a group comprising 16 patients with CaP (metastatic and localized disease) and 15 healthy individuals. MP, combined with multivariate analysis, yielded 17 serum proteins specific to metastatic disease. A single protein detected at m/z 7771 was found to be significantly decreased in the sera of all the metastatic CaP patients but not in localized CaP or healthy individuals. This protein was therefore chosen as the primary candidate for further analysis. The complex nature of the serologic proteome necessitated an isolation strategy that included a C18 prefractionation, followed by multidimensional liquid chromatography and, finally, two-dimensional gel electrophoresis. The separation process was monitored by UV-Vis and matrix-assisted laser desorption/ionization-time of flight MS analysis. This strategy was found to greatly facilitate subsequent MS characterization of the unknown protein, which was identified as platelet factor 4, a chemokine with prothrombolytic and antiangiogenic activities. Confirmation was achieved using both Western blot analysis and enzyme-linked immunosorbent assay. With the growing interest in using MP for patient classification and diagnosis, our approach and its variations should be powerful in the separation and characterization of proteins following MP.     

Facile monitoring of avian reovirus σB expression and purification processes by tagged green fluorescent protein

Avian reovirus capsid protein σB was genetically fused with a histidine (His₆) tag and a UV-optimized green fluorescent protein (GFPuv) and expressed in Sf-9 cells. The fluorescence of GFPuv allowed for easy identification of protein localization and revealed that the fusion protein was quite stable in the cell culture. The fluorescence intensity (FI) exhibited a linear relationship (r² = 0.93) with the recombinant protein yield and therefore allowed for on-line tracking of the expression profile, which revealed an extremely high maximum yield of 70 μg per 10⁶ cells. The recombinant protein was purified via immobilized metal affinity chromatography (IMAC) and a high purity (85%) was achieved in one step. During the purification, the fluorescence again enabled qualitative and quantitative monitoring of when and how much the desired product was eluted. The GFP-tagging strategy eliminated the need for cumbersome and time-consuming assays (e.g. Western blot or ELISA) for product analysis, thus GFP is an effective non-invasive on-line marker for the expression and purification of recombinant proteins in the baculovirus expression system.     

Multidimensional protein identification technology: current status and future prospects

Protein profiling using high-throughput tandem mass spectrometry has become a powerful method for analyzing changes in global protein expression patterns in cells and tissues as a function of developmental, physiologic and disease processes. This review summarizes the utility and practical application of multidimensional protein identification technology as a platform for comprehensive proteomic profiling of complex biologic samples. The strengths and potential problems and limitations associated with this powerful technology are discussed, with an emphasis placed on one of the biggest challenges currently facing large-scale expression profiling projects – namely, data analysis. Complementary bioinformatic computational data mining strategies, such as clustering, functional annotation and statistical inference, are also discussed as these are increasingly necessary for interpreting the results of global proteomic profiling studies.     

Proteomic studies support the use of multi-product immunoassays to monitor host cell protein impurities

In the biopharmaceutical industry, recombinant protein drugs are commonly produced in Chinese hamster ovary (CHO) cells. During the development process, removal of CHO cell-derived proteins from the biopharmaceutical product is monitored using multi-product immunoassays. Such immunoassays are developed by raising antibodies to a single CHO cell protein preparation. However, these assays are utilized to monitor CHO cell protein impurities during the recovery of products from different CHO cell lines. To address whether underlying differences between CHO cell lines result in sufficient protein expression changes to exclude the suitability of multi-product immunoassays, a comparative proteomics study of three independently generated CHO cell lines was performed. Statistical analysis of over 1000 proteins resolved by 2-D PAGE demonstrated that the protein expression profiles of three different CHO cell lines exhibit very few differences in protein expression. Only 11 qualitative changes in protein expression and 26 quantitative changes greater than two-fold were observed. Identification of protein spots by mass spectrometry revealed that many of the observed changes were due to post-translational modifications rather than expression of novel proteins in each cell line. These results suggest that multi-product immunoassays are suitable for monitoring host cell proteins in biopharmaceuticals produced in different CHO cell lines.     

Proteomic analysis of nicotine-associated protein expression in the striatum of repeated nicotine-treated rats

Through the proteomic analysis using 2-dimensional electrophoresis, the nicotine addiction-associated proteins were extensively screened in the striatum of rat brains. The nicotine addiction was developed by repeated nicotine injection (0.4mg/kg s.c.), twice daily for 7 days, followed by one challenge injection after a 3 day withdrawal period, and then confirmed by observing a 2.3-fold increase in locomoter activity. The 3 up- and 4 down-regulated proteins were selected and identified to be zinc-finger binding protein-89 (ZBP-89), 2'3'-cyclic nucleotide 3'-phosphodiesterase 1, deoxyribonuclease 1-like 3 (DNase1l3), tandem pore domain halothane inhibited K(+) channel (THIK-2), brain-specific hyaluronan-binding protein (BRAL-1), death effector domain-containing DNA binding protein (DEDD), and brain-derived neurotrophic factor (BDNF) by mass spectrophotometric fingerprinting. Among them, the expression patterns of ZEB-89, DNase1l3, THIK-2, DEDD, and BDNF mRNAs were found to be coincident with those of cognate proteins, by using RT-PCR analysis. These proteins could be suggested as drug targets to develop a new therapy for nicotine-associated diseases, as well as the clues to understand the mechanism of nicotine.     

Construction of a dual-tag system for gene expression, protein affinity purification and fusion protein processing

An E. coli vector system was constructed which allows the expression of fusion genes via a l-rhamnose-inducible promotor. The corresponding fusion proteins consist of the maltose-binding protein and a His-tag sequence for affinity purification, the Saccharomyces cerevisiae Smt3 protein for protein processing by proteolytic cleavage and the protein of interest. The Smt3 gene was codon-optimized for expression in E. coli. In a second rhamnose-inducible vector, the S. cerevisiae Ulp1 protease gene for processing Smt3 fusion proteins was fused in the same way to maltose-binding protein and His-tag sequence but without the Smt3 gene. The enhanced green fluorescent protein (eGFP) was used as reporter and protein of interest. Both fusion proteins (MalE-6xHis-Smt3-eGFP and MalE-6xHis-Ulp1) were efficiently produced in E. coli and separately purified by amylose resin. After proteolytic cleavage the products were applied to a Ni-NTA column to remove protease and tags. Pure eGFP protein was obtained in the flow-through of the column in a yield of around 35% of the crude cell extract.     

A strategy for high-resolution protein identification in surface-enhanced laser desorption/ionization mass spectrometry: calgranulin A and chaperonin 10 as protein markers for endometrial carcinoma

Surface-enhanced laser desorption/ionization-mass spectrometry (SELDI-MS) has conventionally been practiced on linear time of flight (TOF) which has low mass accuracy and resolution. Here we demonstrate in an examination of both malignant and nonmalignant endometrial tissue homogenates that high mass accuracy and resolution in the MS stage are crucial. Using a commercially available quadrupole/TOF (QqTOF), we were able to resolve two potential cancer markers, subsequently identified off-line as chaperonin 10 and calgranulin A, that differ by 8 Da in mass. Two off-line protein identification protocols were developed: the first was based on size-exclusion chromatography (SEC), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), protein extraction, trypsin digestion, and matrix-assisted laser desorption/ionization-tandem MS (MALDI-MS/MS); the second on SEC and shotgun nano-liquid chromatography (nanoLC)-MS/MS. Analyses on a cohort of 44 endometrial homogenates showed 22 out of 23 nonmalignant samples had nondetectable to very low abundance of chaperonin 10 and calgranulin A; 17 of the 21 malignant samples had detectable to abundant levels of both proteins. Immunohistochemical staining of a tissue microarray of 32 samples showed that approximately half of malignant endometrial tissues exhibited positive staining for calgranulin A in the malignant epithelium, while 9 out of 10 benign tissues exhibited negative epithelial staining. In addition, macrophages/granulocytes in malignant as well as nonmalignant tissues showed positive staining. No immunostaining occurred in stroma or myometrium. Calgranulin A, in combination with chaperonin 10 and other proteins, may eventually constitute a panel of markers to permit diagnosis and screening of endometrial cancer.