Detection and identification of heat shock protein 10 as a biomarker in colorectal cancer by protein profiling

Although colorectal cancer is one of the best-characterized tumors with regard to the multistep progression, it remains one of the most frequent and deadly neoplasms. For a better understanding of the molecular mechanisms behind the process of tumorigenesis and tumor progression, changes in protein expression between microdissected normal and tumorous colonic epithelium were analyzed. Cryostat sections from colorectal tumors, adenoma tissue, and adjacent normal mucosa were laser-microdissected and analyzed using ProteinChip Arrays. The derived MS profiles exhibited numerous statistical differences. One peak showing significantly high expression in the tumor was purified by reverse-phase chromatography and SDS-PAGE. The protein band of interest was passively eluted from the gel and identified as heat shock protein 10 (HSP 10) by tryptic digestion, peptide mapping, and MS/MS analysis. This tumor marker was further characterized by immunohistochemistry. Analysis of HSP 10-positive tissue by ProteinChip technology confirmed the identity of this protein. This work demonstrates that biomarker in colorectal cancer can be detected, identified, and assessed by a proteomic approach comprising tissue microdissection, protein profiling, and immunological techniques. In our experience, histological defined microdissected tissue areas should be used to identify proteins that might be responsible for tumorigenesis.     

Intact protein profiling in breast cancer biomarker discovery: Protein identification issue and the solutions based on 3D protein separation,bottom‐up and top‐down mass spectrometry

Proteomics profiling of intact proteins based on MALDI‐TOF MS and derived platforms has been used in cancer biomarker discovery studies. This approach suffers from a number of limitations such as low resolution, low sensitivity, and that no knowledge is available on the identity of the respective proteins in the discovery mode. Nevertheless, it remains the most high‐throughput, untargeted mode of clinical proteomics studies to date. Here we compare key protein separation and MS techniques available for protein biomarker identification in this type of studies and define reasons of uncertainty in protein peak identity. As a result of critical data analysis, we consider 3D protein separation and identification workflows as optimal procedures. Subsequently, we present a new protocol based on 3D LC‐MS/MS with top‐down at high resolution that enabled the identification of HNRNP A2/B1 intact peptide as correlating with the estrogen receptor expression in breast cancer tissues. Additional development of this general concept toward next generation, top‐down based protein profiling at high resolution is discussed.     

Serum, liver, and kidney proteomic analysis for the alloxan-induced type I diabetic mice after insulin gene transfer of naked plasmid through electroporation

Gene therapy has been reported to be effective in treating diabetes mellitus (DM), while little has been found out about the functional protein changes since. The liver and kidney play important roles in glucose absorption, metabolism, and excretion. Changes in the two organs may reflect pathologic alterations during DM, while the serum has a direct connection with most organs and pathological changes. We used alloxan to induce diabetic mice, electrotranferred the insulin gene into their sural muscles, and discovered that their blood glucose decreased to normal level. Consequently, proteomic approaches were applied to evaluate protein changes in the liver, kidney, and serum of normal, diabetic, and gene transferred mice. Forty-three proteins were found either up-regulated or down-reglulated in the liver, kidney, and serum of the alloxan-induced type I diabetic mice. Only five proteins in the liver, five proteins in the kidney, and seven proteins in the serum of diabetic mice were found to be back-regulated to normal levels after gene transfer. These back-regulated proteins are involved in lipid and glucose metabolism, associated with phosphorylation, signal transduction, oxidation, and immune inflammation. Our findings might promote a better understanding for the mechanism of DM, and provide novel targets for estimating the effects of gene therapy.     

Identification of commercial hake and grenadier species by proteomic analysis of the parvalbumin fraction

Analysis of parvalbumin fractions through proteomic methodologies allowed the differential classification of ten commercial, closely related species of the family Merlucciidae. Muscle extracts from nine hake species of the genus Merluccius including two subspecies of Merluccius australis (australis and polylepsis) and one grenadier species Macruronus novaezelandiae with two populations (novaezelandiae and magellanicus) were evaluated by 2-DE and MALDI-TOF MS. 2-DE demonstrated that the species tested displayed a low intra-specific degree of polymorphism and the isoform patterns were noticeably species-specific. MALDI-TOF mass fingerprints showed clear differences in the pattern of peptides produced by tryptic digestion between the Merluccius and the Macruronus, making the genus differentiation possible. In addition, a selective peptide mass present in the spectra from certain hakes allowed its classification in two groups: Euro-African and American hakes. Besides, some specific masses allowed a clearly individual identification for M. bilinearis, M. australis polylepsis, M. australis australis, M. productus, M. paradoxus and M. polli, while the rest of the hake species can be grouped in two clusters, comprising M. hubbsi and M. gayi in one and M. merluccius and M. capensis in the other.     

N-glycan biosynthesis inhibitors induce in vitro anticancer activity in colorectal cancer cells

During malignant transformation, changes in the expression profile of glycans may be involved in a variety of events, including the loss of cell-cell and cell-matrix adhesion, migration, invasion, and evasion of apoptosis. Therefore, modulation of glycan expression with drugs has promising therapeutic potential for various cancer types. In this study, we investigated the in vitro anticancer activity of the N-glycan biosynthesis inhibitors (swainsonine and tunicamycin) in cells derived from colorectal cancer (CRC). We also examined whether these inhibitors are able to induce radiosensitization and toxicity when used in combination with cisplatin or irinotecan, two current anticancer drugs. Our results show that treatment with tunicamycin inhibits cellular mechanisms related to the malignant phenotype, such as anchorage-dependent and anchorage-independent colony formation, migration and invasion, in undifferentiated HCT-116 colon cancer cells, whereas swainsonine only inhibits cell migration. We also observed that tunicamycin, but not swainsonine, caused radiosensitivity in HCT-116 cells. Moreover, the combination of swainsonine with cisplatin or irinotecan enhanced their toxicity in HCT-116 cells, while the combination of tunicamycin with these drugs had no effect. Given these results, we suggest that the modulation of N-glycan biosynthesis appears to be a potential therapeutic tool for CRC treatment because inhibition of this process induced anticancer activity in vitro. Additionally, the inhibition of the N-glycan biosynthesis in combination with chemotherapic drugs is a promising therapeutic strategy for enhancing radiation therapy.     

Influence of storage conditions on MALDI‐TOF MS profiling of gingival crevicular fluid: Implications on the role of S100A8 and S100A9 for clinical and proteomic based diagnostic investigations

Gingival crevicular fluid (GCF) may be a source of diagnostic biomarkers of periodontitis/gingivitis. However, peptide fingerprints may change, depending on GCF collection, handling and storage. We evaluated how storage conditions affect the quality and the reproducibility of MALDI‐TOF profiles of this fluid. GCF was collected on paper strips from four subjects with healthy gingiva. Our findings demonstrated that sample storage conditions significantly affect GCF peptide pattern over time. Specifically, the storage of GCF immediately extracted from paper strips generates less variations in molecular profiles compared to the extraction performed after the storage. Significant spectral changes were detected for GCF samples stored at –20°C directly on the paper strips and extracted after three months, in comparison to the freshly extracted control. Noteworthy, a significant decrease in the peak area of HNP‐3, S100A8, full‐length S100A9 and its truncated form were detected after 3 months at –80°C. The alterations found in the “stored GCF” profile not only may affect the pattern‐based biomarker discovery but also make its use not adequate for in vitro diagnostic test targeting S100A8, S100A9 proposed as potential diagnostic biomarkers for periodontal disease. In summary, this study shows that the best preserved signatures were obtained for the GCF samples eluted in trifluoroacetic acid and then immediately stored at –80°C for 1 month. The wealth of information gained from our data on protein/patterns stability after storage might be helpful in defining new protocols which enable optimal preservation of GCF specimen.     

Systematical evaluation of the effects of sample collection procedures on low-molecular-weight serum/plasma proteome profiling

Blood is an ideal source for biomarker discovery. However, little has been done to address the effects of sampling, handling and storage procedures on serum/plasma proteomes. We used magnetic bead-based MALDI-TOF MS to systematically evaluate the influence of each procedure on low-molecular-weight serum/plasma proteome profiling on the basis of the whole spectra. We found that sampling procedures, including the selection of blood collection tubes and anticoagulants, variations in clotting time or time lag before centrifugation, and hemolysis, displayed significant effects on the proteomes. Moreover, serum and plasma were mutually incompatible for proteome comparison. By contrast, overnight fasting, handling procedures, including centrifugation speeds (1500 x g vs. 3000 x g) or time (15 min vs. 30 min), and storage conditions, such as at 4 degrees C or 25 degrees C for up to 24 h or at -80 degrees C for up to 3 months, and repeated freeze/thaw of up to ten cycles, had relatively minor effects on the proteomes based upon our analysis of about 100 peaks. We concluded that low-molecular-weight serum/plasma proteomes were diversely affected by sampling, handling and storage with most change from variations of sampling procedures. We therefore suggest the necessity of standardizing sampling procedure for proteome comparison and biomarker discovery.     

Toward the proteomic identification of biomarkers for the prediction of HBV related hepatocellular carcinoma

Early detection is a key step for effective intervention of hepatocellular carcinoma (HCC), the lack of sensitive and specific biomarkers is a major reason for the high rate of HCC-related mortality. This report described an integrated strategy by combining SELDI-ProteinChip, sophisticated algorithm analysis, acetonitrile (ACN) pre-treatment and two-dimensional electrophoresis (2DE)-peptide mass fingerprinting (PMF) techniques to identify serological markers for the prediction of HBV-related HCC. Proteomic profiling of three groups of serum specimens from HBV-related HCC (50 cases), HBV infection (45 cases), and normal subjects (30 cases) was conducted by using SELDI-ProteinChip system and the resulting different protein peaks were subjected to stepwise statistical analyses. Three most discriminatory peaks at 5890, 11615, and 11724 Da, respectively, were screened out from the statistical algorithm and a predictive model based on the three peaks was constructed and tested using the newly enrolled serum samples. 2DE was applied to separate and compare the serum samples that were pre-treated by ACN precipitation. The protein spots obviously intensified in HCC sera in the 2DE region of 12 kDa were identified by PMF to be serum SAA, which was validated by SELDI-TOF spectra of HCC sera after immunoprecipitation using anti-SAA antibody and by Western blot experiments. Given the fact that SAA is not a specific biomarker, further attempt is being made to identify the other two most discriminatory peaks to realize the possibility of using the predictive model for HCC surveillance and prediction.     

The proteomic alterations of Thermoanaerobacter tengcongensis cultured at different temperatures

Thermoanaerobacter tengcongensis, one of many thermophilic organisms, survives harsh living conditions in temperatures ranging from 50 to 80 degrees C. In this comprehensive analysis, we present a robust approach, 2-DE and MALDI-TOF MS, to compare and identify the bacterial proteins responding to the temperature stress. In total, 164 spots of 2-DE were found with the significant changes in spot volume at three culture temperatures, 55, 75, and 80 degrees C, respectively; furthermore, 87 unique proteins were characterized by MS. Our results reveal that the electrophoretic images of the bacterial proteins, extracted from two culture temperatures (55 and 75 degrees C), had similar patterns; however, the bacteria cultured at 80 degrees C had dramatically decreased their spot volumes. Additionally, the temperature-sensitive proteins are broadly divided into two groups: specific expression at certain temperatures and consistent changes of expression responsive to temperature. For instance, three proteins closely related with redox regulation, dihydrolipoamide acyltransferase, NADH:ubiquinone oxidoreductase, and ferredoxin, were only detected in the bacteria cultured at 55 degrees C. Whereas, two chaperonins, GroES and GroEL, were found to show a consistent increase during the elevated temperatures with the determinations, either by MS or Western blot. The proteomic information, thus expedites our understanding of the molecular mechanisms regarding how thermophilic bacteria adapt to the alterations in living environment.     

Detection of hydrolysis of lipid post-translational modifications during gel-electrophoresis-based proteomic protocol

The influence of sample preparation on the identification of a lipid PTM was examined. Nonspecific lipid transfer protein 1 (LTP1) from barley is modified with a lipid-like molecule of mass of 294 Da. This modification was detected in the MS analysis of intact protein samples but no lipid-bound peptide was observed in the MS analysis of the in-gel digested LTP1 after an SDS-PAGE separation of the protein mixture. By using SEC instead of SDS-PAGE, the lipid-modified peptide was observed after in-solution enzymatic digestion of the SEC fraction containing LTP1. Conditions of individual steps of the gel-electrophoresis-based protocol were tested to find their effect on the removal of the lipid PTM from LTP1. The influences of particular solutions used in the gel-electrophoresis-based protocol on the hydrolysis of lipids were investigated. It was found that denaturing conditions, in combination with alkaline pH, have a major influence on the hydrolysis of the ester bond. Especially, the electrophoretic buffer has a strong influence on the hydrolysis of the lipid PTM (in the intact molecule) of LTP1.     

Proteomic changes in hearts of kyphoscoliosis (ky) mutant mice in the absence of structural pathology: implication for the analysis of early human heart disease

Complex molecular changes associated with early stage human heart disease are poorly understood and prevent the development of effective treatments of human cardiac disease. Relatively minor structural changes in early disease may accompany some conditions such as arrhythmias. Our objective was to determine if significant proteomic changes occur in heart tissues in the absence of structural pathology. We used a proteomic "pipeline" based on Ciphergen SELDI-TOF/MS, gel electrophoresis and MALDI-TOF/MS. The kyphoscoliosis (ky) mouse carries a mutation in a putative transglutaminase causing a primary skeletal muscle disease. The ky protein is expressed usually in skeletal and cardiac muscle but its absence from the ky heart causes no structural pathology making it a good model of "occult" heart disease. We discovered 20 statistically validated biomarkers discriminating ky from normal hearts, one cardiac troponin-I was reduced by 40% in ky hearts. A 17% deficit was confirmed subsequently by Western blot. Thus, the proteome of ky hearts was abnormal, giving support to our contention that this SELDI-based analytical approach is capable of making a significant contribution to the analysis of complex proteomic changes in early stage human heart disease.     

Differential proteomic analysis of proteins in wheat spikes induced by Fusarium graminearum

Scab, caused by Fusarium graminearum, is a serious spike disease in wheat. To identify proteins in resistant wheat cultivar Wangshuibai induced by F. graminearum infection, proteins extracted from spikes 6, 12 and 24 h after inoculation were separated by 2-DE. Thirty protein spots showing 3-fold change in abundance when compared with treatment without inoculation were characterized by MALDI-TOF MS and matched to proteins by querying the mass spectra in protein databases or the Triticeae EST translation database. Based on their volume profiles, these proteins were classified into four categories. The first one fell off rapidly at the initial inoculation and then rose at 12 or 24 hai, the second one decreased considerably after inoculation and remained at low level, the third one rose at the initial inoculation and then declined at 12 or 24 hai, the forth one showed steady increase after inoculation and maintained at a high level. Many of the proteins identified in the first two categories are related to carbon metabolism and photosynthesis. While most of proteins identified in the last two categories are related to stress defense of plants, indicating that proteins associated with the defense reactions were activated or translated shortly after inoculation.     

Evaluation of VITEK matrix‐assisted laser desorption ionization–time of flight mass spectrometry for identification of anaerobes

Rapid and adequate identification of anaerobic bacterial species still presents a challenge for most diagnostic laboratories, hindering the selection of appropriate therapy. In this study, the identification capacity of 16S rRNA sequence analysis, VITEK 2 (BioMérieux, Lyon, France) compact analysis and VITEK MS‐mediated identification for anaerobic bacterial species was compared. Eighty‐five anaerobic bacterial isolates from 11 provinces in China belonging to 14 genera were identified by these three methods. Differences in identification between these three methods were compared. Consistent identification results were obtained for 54 (54/85, 63.5%) isolates by all three methods, the most discordant results being concentrated in Clostridium XI (n = 8) and Bacteroides fragilis (n = 9) clusters. Using the VITEK MS system, 74 (74/90, 82.2%) isolates were identified as single species consistent with 16S rRNA sequence analysis, which was significantly better than the results obtained with VITEK 2 Compact (P < 0.01). Misidentifications by the Vitek 2 Compact and Vitek MS systems were mainly observed in the Clostridium XI (n = 8)and B. fragilis clusters (n = 9). VITEK MS identified anaerobic bacteria even after they had been exposed to oxygen for a week. Identification by the Vitek MS system was more consistent with 16S rRNA sequence analysis than identification by Vitek 2 Compact. Continuous expansion of the VITEK MS database with rare described anaerobic species is warranted to improve both the efficiency and accuracy of VITEK MS identification in routine diagnostic microbiology.     

Proteomic profiling of Cronobacter turicensis 3032, a food‐borne opportunistic pathogen

Members of the genus Cronobacter are opportunistic pathogens for neonates and are often associated with contaminated milk powder formulas. At present little is known about the virulence mechanisms or the natural reservoir of these organisms. The proteome of Cronobacter turicensis 3032, which has recently caused two deaths, was mapped aiming at a better understanding of physiology and putative pathogenic traits of this clinical isolate. Our analyses of extracellular, surface‐associated and whole‐cell proteins by two complementary proteomics approaches, 1D‐SDS‐PAGE combined with LC‐ESI‐MS/MS and 2D‐LC‐MALDI‐TOF/TOF MS, lead to the identification of 832 proteins corresponding to a remarkable 19% of the theoretically expressed protein complement of C. turicensis. The majority of the identified proteins are involved in central metabolic pathways, translation, protein folding and stability. Several putative virulence factors, whose expressions were confirmed by phenotypic assays, could be identified: a macrophage infectivity potentiator involved in C. turicensis persistence in host cells, a superoxide dismutase protecting the pathogen against reactive oxygen species and an enterobactin‐receptor protein for the uptake of siderophore‐bound iron. Most interestingly, a chitinase and a metalloprotease that might act against insects and fungi but no casein hydrolysing enzymes were found, suggesting that there is an environmental natural habitat of C. turicensis 3032.     

Proteomic analysis of the effects of baicalein on colorectal cancer cells

Baicalein is the flavonoids with multiple pharmacological activities. The aim of our study was to investigate the effects of baicalein on colorectal cancer (CRC) and to recognize the targets of baicalein treatment. To better understand baicalein's target, proteomic approaches were used to purify and identify the protein substrates using 2D difference gel electrophoresis (2D SDS-PAGE) to elucidate proteins differential display. Results from this study investigate that baicalein treatment of CRC cells results in reduced cell proliferation. As a result, differential protein displays between baicalein-treated and untreated CRC were determined and validated. There were 11 differentially expressed proteins between baicalein-treated and untreated CRC. Furthermore, we demonstrate that baicalein inhibits cancer cell proliferation and reduced reactive oxygen species (ROS) by up-regulating the levels of peroxiredoxin-6 (PRDX6). Knockdown of PRDX6 in baicalein-treated CRC cells by specific small interfering RNA resulted in ROS production and proliferation, opposite of the baicalein treatment scenario as indicated by cell cycle distribution. These results illustrate that baicalein up-regulates the expression of PRDX6, which attenuates the generation of ROS and inhibits the growth of CRC cells, whereas baicalein treatment have no effect on normal epithelial cells.     

Proteomic analysis of cold adaptation in a Siberian permafrost bacterium--Exiguobacterium sibiricum 255-15 by two-dimensional liquid separation coupled with mass spectrometry

Bacterial cold adaptation in Exiguobacterium sibiricum 255-15 was studied on a proteomic scale using a 2-D liquid phase separation coupled with MS technology. Whole-cell lysates of E. sibiricum 255-15 grown at 4 degrees C and 25 degrees C were first fractionated according to pI by chromatofocusing (CF), and further separated based on hydrophobicity by nonporous silica RP HPLC (NPS-RP-HPLC) which was on-line coupled with an ESI-TOF MS for intact protein M(r) measurement and quantitative interlysate comparison. Mass maps were created to visualize the differences in protein expression between different growth temperatures. The differentially expressed proteins were then identified by PMF using a MALDI-TOF MS and peptide sequencing by MS/MS with a MALDI quadrupole IT TOF mass spectrometer (MALDI-QIT-TOF MS). A total of over 500 proteins were detected in this study, of which 256 were identified. Among these proteins 39 were cold acclimation proteins (Caps) that were preferentially or uniquely expressed at 4 degrees C and three were homologous cold shock proteins (Csps). The homologous Csps were found to be similarly expressed at 4 degrees C and 25 degrees C, where these three homologous Csps represent about 10% of the total soluble proteins at both 4 degrees C and 25 degrees C.     

Peptidome profiling of induced sputum by mesoporous silica beads and MALDI-TOF MS for non-invasive biomarker discovery of chronic inflammatory lung diseases

Induced sputum is recognized as being of increasing importance for the diagnosis and monitoring of chronic inflammatory lung diseases. The main purpose of this study is to provide a valid approach to better fractionate and characterize the still under-estimated low-molecular weight proteome of induced sputum by using mesoporous silica beads (MSBs) SPE coupled to MALDI-TOF MS. Sputum peptides were captured from both derivatized and non-derivatized MSBs and then profiled by MALDI-TOF MS. Depending on the chemical groups present on the mesoporous surface, complex peptide mixtures were extracted from induced sputum and converted into reproducible MALDI profiles. The number of peaks detected as a function of S/N was evaluated for each mesoporous surface. More than 400 peaks with an S/N>5 were obtained in comparison to 200 peaks detected without MSBs. Additionally, as a proof-of-principle, we investigated the ability of this platform to discriminate between the "sputome" of patients with asthma and chronic obstructive pulmonary disease, and between these groups and those of healthy control subjects. Six m/z peaks emerged as potential diagnostic peptidic patterns able to differentiate these inflammatory airway diseases in the sputome range. Human α-defensins (human neutrophil peptide (HNP)1, HNP2, HNP3) and three C-terminal amidated peptides, one of which is phosphorylated on serine, were identified by MALDI-TOF/TOF MS. These findings may contribute to defining a high-throughput screening MS-based platform for monitoring key peptidic-biomarkers for inflammatory and chronic respiratory diseases in induced sputum samples.     

Proteome mapping of the Drosophila melanogaster male reproductive system

The fruit fly Drosophila melanogaster is an excellent model organism for studying insect reproductive biology. Although the gene expression profiles of both male and female reproductive organs have been studied in detail, their proteomic profiles and functional characteristics largely remained to be clarified. In this study, we conducted proteome mapping of the male internal reproductive organs using 2‐DE. We identified a total of 440 protein components from gels of the male reproductive organs (testis, seminal vesicle, accessory gland, ejaculatory duct, and ejaculatory bulb). A number of proteins associated with odorant/pheromone‐binding, lipid metabolism, proteolysis, and antioxidation were expressed tissue specifically in the male reproductive system. Based on our proteomic data set, we constructed reference proteome maps of the reproductive organs, which will provide valuable information toward a comprehensive understanding of Drosophila reproduction.