Glycoproteomic analysis and molecular modeling of haptoglobin multimers.

Extra-thiol groups on the α-subunit allow haptoglobin (Hp) to form a variety of native multimers which influence the biophysical and biological properties of Hp. In this work, we demonstrated how differences of multimeric conformation alter the glycosylation of Hp. The isoform distributions of different multimers were examined by an alternative approach, i.e. 3-D-(Native/IEF/SDS)-PAGE, which revealed differences in N-glycosylation among individual multimers of the same Hp sample. Glycomic mapping of permethylated N-glycan indicated that the assembled monomer and multimeric conformation modulate the degree of glycosylation, especially the reduction in terminal sialic acid residues on the bi-antennary glycan. Loss of the terminal sialic acid in the higher order multimers increases the number of terminal galactose residues, which may contribute to conformation of Hp. A molecular model of the glycosylated Hp multimer was constructed, suggesting that the effect of steric hindrance on multimeric formation is critical for the enlargement of the glycan moieties on either side of the monomer. In addition, N241 of Hp was partially glycosylated, even though this site is unaffected by steric consideration. Thus, the present study provides evidence for the alteration of glycan structures on different multimeric conformations of Hp, improving our knowledge of conformation-dependent function of this glycoprotein.

Glycoproteomics analysis to identify a glycoform on haptoglobin associated with lung cancer.

Glycosylation is a common protein modification that is of interest in current cancer research because altered carbohydrate moieties are often found during cancer progress. A search for biomarkers in human lung cancer serum samples using glycoproteomic approaches identified fucosylated haptoglobin (Hp) significantly increased in serum of each subtype of lung cancer compared to normal donors. In addition, MS provided evidence of an increase of Hp fucosylation; the glycan structure was determined to be an α 2,6-linked tri-sialylated triantennary glycan containing α1,3-linked fucose attached to the four-linked position of the three-arm mannose of N-linked core pentasaccharide. These preliminary findings suggest that the specific glycoform of Hp may be useful as a marker to monitor lung cancer progression.

Different sample preparation and detection methods for normal and lung cancer urinary proteome analysis.

The urinary proteome is known to be a valuable field of study related to human physiological functions because many components in urine provide an alternative to blood plasma as a potential source of disease biomarkers useful in clinical diagnosis and therapeutic application. Due to the variability and complexity of urine, sample preparation is very important for decreasing the dynamic range of components and isolating specific urinary proteins prior to analysis. We discuss many useful sample preparation methods in this chapter, including those of lung cancer urine samples. In addition, protein detection methods are also crucial in visualizing protein profiles and for quantification of protein content in urine samples from both normal donor and lung cancer patients. This chapter also provides alternative choices of urine sample preparation and detection methods for selective use in urinary proteome analysis and for identifying urinary protein markers in lung cancer and other diseases.

Glycoproteomic analysis of WGA-bound glycoprotein biomarkers in sera from patients with lung adenocarcinoma.

Differential protein expression profiles in the serum samples from patients with lung adenocarcinoma may be associated with glycosylation during cancer development. In this study, we used various glycoproteomic approaches to investigate the different glycoproteomic profiles of human normal and lung adenocarcinoma serum samples and to investigate putative altered glycoprotein biomarkers. In our preliminary screening, FITC-labeled lectin staining was used for the detection of specific glycoprotein profiles. wheat germ agglutinin (WGA) lectin had the highest level of specific binding to glycoproteins in both samples. We enriched for glycoproteins in the serum samples using WGA lectin affinity and then performed co-immunoprecipitation with anti-haptoglobin and 2-DE, 2-D difference in-gel electrophoresis and MS analyses. From these analyses, we identified 39 differentially expressed proteins, including 27 up-regulated proteins and 12 down-regulated proteins. Bioinformatics tools were used to search for protein ontology, category classifications and prediction of glycosylation sites. In addition, three up-regulated glycoproteins (adiponectin, cerulolasmin and glycosylphosphatidyl-inositol-80) and two down-regulated glycoproteins (cyclin H and Fyn) that were found to be correlated with lung cancer development were validated by Western blot analysis. We suggest that these altered glycoproteins may be useful as biomarkers for lung cancer development and progression.

Targeting the delivery of glycan-based paclitaxel prodrugs to cancer cells via glucose transporters.

This report describes the synthesis of four novel paclitaxel based prodrugs with glycan conjugation (1-4). Glycans were conjugated using an ester or ether bond as the linker between 2'-paclitaxel and the 2'-glucose or glucuronic acid moiety. These prodrugs showed good water solubility and selective cytotoxicity against cancer cell lines, but showed reduced toxicity toward normal cell lines and cancer cell lines with low expression levels of GLUTs. The ester conjugated prodrug 1 showed the most cytotoxicity among the prodrugs examined and could be transported into cells via GLUTs. Fluorescent and confocal microscopy demonstrated that targeted cells exhibited morphological changes in tubulin and chromosomal alterations that were similar to those observed with paclitaxel treatment. Therefore, these glycan-based prodrugs may be good drug candidates for cancer therapy, and the glycan conjugation approach is an alternative method to enhance the targeted delivery of other drugs to cancer cells that overexpress GLUTs.

Current proteomic analysis and post-translational modifications of biomarkers in human lung cancer materials.

Lung cancer is the leading cause of cancer deaths worldwide and is often diagnosed in the advanced or late stages. The discovery of candidate biomarkers in human lung cancer materials holds clinical potential as well as a significant challenge. Due to a large number of proteins in human materials that form the protein complexity and post-translational modifications (PTMs) of proteins, proteomic analysis of lung cancer biomarkers remains a difficult task. Many proteomic approaches have been used to investigate the biomarkers in human lung cancer materials. In this review, we summarize the current proteomic approaches employed for analysis of proteome profiles in human lung cancer materials and some PTMs causing different proteome profiles and expression levels of biomarkers in lung cancer. In addition, bioinformatic tools for PTMs are useful for prediction of glycosylation and phosphorylation sites in proteins, which can help us further understand the correlation of PTMs and tumor development. However, it is necessary that proteome analysis of lung cancer materials be incorporated with new strategies and technologies to improve the efficiency of the analysis and to investigate novel biomarkers that would be very useful for further diagnostic and therapeutic applications.

Enhanced detectability in proteome studies.

The discovery of candidate biomarkers from biological materials coupled with the development of detection methods holds both incredible clinical potential as well as significant challenges. However, the proteomic techniques still provide the low dynamic range of protein detection at lower abundances. This review describes the current development of potential methods to enhance the detection and quantification in proteome studies. It also includes the bioinformatics tools that are helpfully used for data mining of protein ontology. Therefore, we believe that this review provided many proteomic approaches, which would be very potent and useful for proteome studies and for further diagnostic and therapeutic applications.

Functional proteomics and correlated signaling pathway of the thermophilic bacterium Bacillus stearothermophilus TLS33 under cold-shock stress.

The thermophilic bacterium Bacillus stearothermophilus TLS33 was examined under cold-shock stress by a proteomic approach to gain a better understanding of the protein synthesis and complex regulatory pathways of bacterial adaptation. After downshift in the temperature from 65 degrees C, the optimal growth temperature for this bacterium, to 37 degrees C and 25 degrees C for 2 h, we used the high-throughput techniques of proteomic analysis combining 2-DE and MS to identify 53 individual proteins including differentially expressed proteins. The bioinformatics database was used to search the biological functions of proteins and correlate these with gene homology and metabolic pathways in cell protection and adaptation. Eight cold-shock-induced proteins were shown to have markedly different protein expression: glucosyltransferase, anti-sigma B (sigma(B)) factor, Mrp protein homolog, dihydroorthase, hypothetical transcriptional regulator in FeuA-SigW intergenic region, RibT protein, phosphoadenosine phosphosulfate reductase and prespore-specific transcriptional activator RsfA. Interestingly, six of these cold-shock-induced proteins are correlated with the signal transduction pathway of bacterial sporulation. This study aims to provide a better understanding of the functional adaptation of this bacterium to environmental cold-shock stress.

Proteomics viewed on stress response of thermophilic bacterium Bacillus stearothermophilus TLS33.

Thermophilic bacterium Bacillus stearothermophilus TLS33, isolated from a hot spring in Chiang Mai, Thailand, usually produces many enzymes that are very useful for industrial applications. However, the functional properties and mechanisms of this bacterium under stress conditions are rarely reported and still need more understanding on how the bacterium can survive in stress environments. In this study, we examined the oxidative stress induced proteins of this bacterium by proteomic approach combining two-dimensional electrophoresis and mass spectrometry. When the bacterium encountered oxidative stress, peroxiredoxin, as an antioxidant enzyme, is one of the interesting stressed proteins which appeared to be systematically increased with different pI. There are four isoforms of peroxiredoxin, denoted as Prx I, Prx II, Prx III and Prx IV, which are observed at the same molecular weight of 27 kDa but differ in pI values of 5.0, 4.87, 4.81 and 4.79, respectively. The H2O2 concentration directly increased Prx II, Prx III and Prx IV intensities, but decreased Prx I intensity. These shifting of peroxiredoxin isoforms may occur by a post-translational modification. Otherwise, the longer time of oxidative stress had not affected the expression level of peroxiredoxin isoforms. Therefore, this finding of peroxiredoxin intends to know the bacterial adaptation under oxidative stress. Otherwise, this protein plays an important role in many physiological processes and able to use in the industrial applications.

Different techniques for urinary protein analysis of normal and lung cancer patients.

Many components in urine are useful in clinical diagnosis and urinary proteins are known as important components to define many diseases such as proteinuria, kidney, bladder and urinary tract diseases. In this study, we focused on the comparison of different sample preparation methods for isolating urinary proteins prior to protein analysis of pooled healthy and lung cancer patient samples. Selective method was used for preliminary investigation of some putative urinary protein markers. Urine samples were passed first through a gel filtration column (PD-10 desalting column) to remove high salts and subsequently concentrated. Remaining interferences were removed by ultrafiltration or four precipitation methods. The analysis of urinary proteins by high-performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed many similarities in profiles among preparation methods and a few profiles were different between normal and lung cancer patients. In contrast, the results of two-dimensional gel electrophoresis (2-DE) showed more distinctly different protein patterns. Our finding showed that the sequential preparation of urinary proteins by gel filtration and ultrafiltration could retain most urinary proteins which demonstrated the highest protein spots on 2-D gels and able to identify preliminary urinary protein markers related to cancer. Although sequential preparation of urine samples by gel filtration and protein precipitation resulted in low amounts of proteins on 2-D gels, high Mr proteins were easily detected. Therefore, there are alternative choices for urine sample preparation for studying the urinary proteome and identifying urinary protein markers important for further preclinical diagnostic and therapeutic applications.

Analysis of lectin-bound glycoproteins in snake venom from the Elapidae and Viperidae families.

This paper describes an efficient method of studying the glycoproteins found in snake venom. The glycosylation profiles of the Elapidae and Viperidae snake families were analyzed using FITC-labeled lectin glycoconjugates. The Con A-agarose affinity enrichment technique was used to fractionate glycoproteins from the N. naja kaouthia venom. The results revealed a large number of Con A binding glycoproteins, most of which have moderate to high molecular weights. To identify the proteins, the isolated glycoprotein fractions were subjected to two-dimensional electrophoresis and MALDI-TOF MS. Protein sequences were compared with published protein databases to determine for their biological functions.

Capillary electrophoresis using immobilized whole cells with overexpressed endothelin receptor for specific ligand screening.

A new capillary electrophoresis method using immobilized cells as the stationary phase has been developed. The power of this method is demonstrated by the separation and identification of endothelin antagonists on a capillary column coated by the transfected Chinese hamster ovary (CHO) cells with overexpressing endothelin receptors. The screening results are validated by functional assays suppressing the increase of intracellular calcium concentration induced by endothelin-1. Instead of making efforts in isolating protein receptors, the easily prepared whole-cell capillary column provides a superior tool on the basis of ligand/receptor affinity for a rapid screening of potent drug candidates from compound libraries.

Proteomics of snake venoms from Elapidae and Viperidae families by multidimensional chromatographic methods.

Snake venoms contain a large number of biologically active substances and the venom components are very useful for pharmaceutical applications. Our goal is to separate and identify components of snake venoms in ten snake species from the Elapidae and Viperidae families using multidimensional chromatographic methods. The multidimensional chromatographic methods include reversed-phase high-performance liquid chromatography (RP-HPLC), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), lab-on-a-chip, two-dimensional electrophoresis (2-DE), and mass spectrometry. The venoms of eight snake species demonstrated major differences in hydrophobicity, molecular weight separations, and 2-DE protein distribution patterns. The 2-DE images showed major differences between families, within each family and even between the same species. Venoms of the Elapidae family showed many basic proteins with a wide range of molecular weights, while venoms of the Viperidae family showed wide ranges of pI and molecular weights, especially for Trimeresurus sp. The multidimensional chromatographic methods revealed specific differences in venom proteins intra-species as well as between species and families. We have isolated and identified proteins that may be unique for each species for further studies in the proteome of snake venoms and their potentially use in the pharmaceutical applications.

Proteomic analysis of a thermostable superoxide dismutase from Bacillus stearothermophilus TLS33.

Thermophilic bacterium Bacillus stearothermophilus TLS33 isolated from a hot spring in Chiang Mai, Thailand produces an extracellular superoxide dismutase (SOD). SOD is a free radical metabolizing enzyme that protects the cell membrane from damage by the highly reactive superoxide free radicals. To identify the secreted SOD, we used the systematically proteomic approaches of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) analysis and database searching. The bacterium was grown in a medium containing 0.1% w/v yeast extract and 0.1% w/v tryptone in 100% v/v base mixture at 65 degrees C for 72 h, by assessing their growth by protein and SOD activity. The bacterium produced the highest SOD activity at 65 degrees C for 48 h and the extracellular SOD was run on 2-D PAGE using broad range pH 3-10 immobilized pH gradients (IPGs) and narrow range pH 4-7 IPGs. The isoelectric point and molecular mass of the extracellular SOD were approximately 5.8 and 28 kDa, respectively. In addition, the NH(2)-terminal amino acid sequence was found to be P-F-E-L-P-A-L-P-Y-P-Y-D-A-L-E-P-P-I-I-D, which had a homology of approximately 85% to the Mn-SOD family and 65% to the Fe-SOD family.

Proteomic study of cold shock protein in Bacillus stearothermophilus P1: Comparison of temperature downshifts.

The thermophilic bacterium Bacillus stearothermophilus P1 is unique in its ability to thrive in extreme environments such as high temperatures or high pH conditions. The study of cold shock response is very interesting and interpreted as a shock response to express the genes involved in synthesis of specific proteins. This study investigated the study of cold shock protein of B. stearothermophilus P1 when the cell culture temperature shifted from 65 degrees C to 37 degrees C and 25 degrees C. Cell growth at 37 degrees C weakly increased in the previous 3 h and then slowly decreased. In contrast, cell growth at 25 degrees C was slowly decreased. The protein contents after temperature downshifts were analyzed by proteomic techniques using protein chip and two-dimensional (2-D) electrophoresis that are highly effective and useful for protein separation and identification. The different proteins after a temperature decrease from 65 degrees C to 37 degrees C and 25 degrees C were expressed on 2-D gel patterns and the cold shock protein was detected in the acidic area with the isoelectric point and molecular mass approximately 4.5 and 7.3 kDa, respectively. The NH(2)-terminal sequence of a major cold shock protein from B. stearothermophilus P1 was MQRGKVKWFNNEKGFGFIEVEGGSD, similar to other cold shock proteins from Bacillus sp. up to 96% identity, but different from the other bacteria with homology less than 80% identity.

Bioinformatics, functional genomics, and proteomics study of Bacillus sp.

The ability of bioinformatics to characterize genomic and proteomic sequences from bacteria Bacillus sp. for prediction of genes and proteins has been evaluated. Genomics coupling with proteomics, which is relied on integration of the significant advances recently achieved in two-dimensional (2-D) electrophoretic separation of proteins and mass spectrometry (MS), are now important and high throughput techniques for qualifying and analyzing gene and protein expression, discovering new gene or protein products, and understanding of gene and protein functions including post-genomic study. In addition, the bioinformatics of Bacillus sp. is embraced into many databases that will facilitate to rapidly search the information of Bacillus sp. in both genomics and proteomics. It is also possible to highlight sites for post-translational modifications based on the specific protein sequence motifs that play important roles in the structure, activity and compartmentalization of proteins. Moreover, the secreted proteins from Bacillus sp. are interesting and widely used in many applications especially biomedical applications that are the highly advantages for their potential therapeutic values.

Expression, purification, crystallization and preliminary crystallographic analysis of a thermostable lipase from Bacillus stearothermophilus P1.

The gene encoding a thermostable lipase secreted by Bacillus stearothermophilus P1 has been cloned and overexpressed in Escherichia coli. The recombinant lipase was purified to homogeneity using ammonium sulfate precipitation, anion-exchange chromatography (Poros 20 HQ) and Sephacryl S-200HR. The molecular mass was shown to be 43 209 Da by mass spectrometry. Crystals suitable for X-ray diffraction analysis were obtained by the hanging-drop method of vapour diffusion with ammonium sulfate as the precipitating agent. Determination of the structure by molecular replacement with existing mesophilic lipase structures has proved unrewarding, as there is less than 20% sequence identity with known lipase structures, but preliminary results with heavy-atom soaking indicate that this strategy will allow the structure to be solved. The availability of this new lipase structure will be of particular significance because it will be the first thermostable lipase to be described.