Protein profile of human hepatocarcinoma cell line SMMC-7721:Identification and functional analysis

AIM： TO investigate the protein profile of human hepatocarcinoma cell line SMMC-7721, to analyze the specific functions of abundant expressed proteins in the processes of hepatocarcinoma genesis, growth and metastasis, to identify the hepatocarcinoma-specific biomarkers for the early prediction in diagnosis, and to explore the new drug targets for liver cancer therapy. METHODS： Total proteins from human hepatocarcinoma cell line SMMC-7721 were separated by two-dimensional electrophoresis （2DE）. The silver-stained gel was analyzed by 2DE software Image Master 2D Elite. Interesting protein spots were identified by peptide mass fingerprinting based on matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry （MALDI-TOF-MS） and database searching. RESULTS： We obtained protein profile of human hepatocarcinoma cell line SMMC-7721. Among the twenty-one successfully identified proteins, mitofilin, endoplasmic reticulum protein ERp29, ubiquinol-cytochrome C reductase complex core protein I, peroxisomal enoyl CoA hydratase, peroxiredoxin-4 and probable 3-oxoacid CoA transferase 1 precursor were the six novel proteins identified in human hepatocarcinoma cells or tissues. Specific functions of the identified heat-shock proteins were analyzed in detail, and the results suggested that these proteins might promote tumorigenesis via inhibiting cell death induced by several cancer-related stresses or via inhibiting apoptosis at multiple points in the apoptotic signal pathway. Other identified chaperones and cancer-related proteins were also analyzed.CONCLUSION： Based on the protein profile of SMMC-7721 cells, functional analysis suggests that the identified chaperones and cancer-related proteins have their own pathways to contribute to the tumorigenesis, tumor growth and metastasis of liver cancer. Furthermore, proteomic analysis is indicated to be feasible in the cancer study.

Polypeptide analysis for nanopore-based protein identification

Presently,proteins are identified by cleaving them with proteases,measuring the mass to charge ratio of the fragments with a mass spectrometer,and matching the fragments to segments within known proteins in databases.We earlier demonstrated that a nanometer-scale pore formed by aerolysin(AeL)can discriminate between,and therefore identify,three similar size proteins from their trypsin-cleaved polypeptide fragments.With this nanopore-protease method,the protein’s identity is instead determined from characteristic ionic current blockade patterns caused by the polypeptide fragments that enter the nanopore.The results also suggested that not all of the theoretically expected cleavage products partition into the pore.To better understand the mechanism by which polypeptide fragments are captured,and how different polypeptides reduce the pore’s ionic current,we studied the effects of 11 identical length polypeptides with different net charges and charge distributions.We show that under certain experimental conditions,negative,positive,and neutral polypeptides are driven into the AeL pore by the same applied voltage polarity.The capture rate and dwell time of polypeptides in the pore depend strongly on the ionic strength,the magnitude of the applied voltage,and the net charge and charge distribution of the polypeptides.The dwell time distribution depends nonmonotonically on the applied voltage(regardless of the polymer’s net charge),and its maximum value depends on the polypeptide net charge and charge distribution.The maximum dwell time for different polypeptides does not occur at the same applied voltage amplitude,which conceivably might complicate the detection and discrimination of some polypeptide fragments.Although additional experiments,computer simulations,and artificial intelligence research are needed to better understand how to optimize the partitioning of enzymatically cleaved fragments into the AeL nanopore,the method is still capable of accurately identifying proteins.

Whole-Cell Protein Identification Using the Concept of Unique Peptides

A concept of unique peptides （CUP） was proposed and implemented to identify whole-cell proteins from tandem mass spectrometry （MS/MS） ion spectra. A unique peptide is defined as a peptide, irrespective of its length, that exists only in one protein of a proteome of interest, despite the fact that this peptide may appear more than once in the same protein. Integrating CUP, a two-step whole-cell protein identification strategy was developed to further increase the confidence of identified proteins. A dataset containing 40,243 MS/MS ion spectra of Saccharomyces cerevisiae and protein identification tools including Mascot and SEQUEST were used to illustrate the proposed concept and strategy. Without implementing CUP, the proteins identified by SEQUEST are 2.26 fold of those identified by Mascot. When CUP was applied, the proteins beating unique peptides identified by SEQUEST are 3.89 fold of those identified by Mascot. By cross-comparing two sets of identified proteins, only 89 common proteins derived from CUP were found. The key discrepancy between identified proteins was resulted from the filtering criteria employed by each protein identification tool. According to the origin of peptides classified by CUP and the commonality of proteins recognized by protein identification tools, all identified proteins were cross-compared, resulting in four groups of proteins possessing different levels of assigned confidence.

The Preparation and Identification of Fumitremorgin B-Hemisuccinate-Carrier Proteins

A method for the preparation and identification of fumitremorgin B-hemisuccinatecarrier protein is described. The overall yield of the fumitremorgin B-hemisuccinate (FTBS) after final purification was 78.6%. The FTBS was characterized by UV, IR, EIMS,element analysis, and 1H, 13C NMR. IR was also used to determine the formation of complete antigen complexes. The reaction route was analysized.

Isolation,identification,and antagonism protein analysis of B34 against Thanatephorus cuc-umeris

A strain B34 against Thanatephorus cucumeris was screened from rice plants. Lab and field experiments showed that the control effects of this fungal strain were better than that of Jinggangmycin on PDA plate. Based on the chemical components of cell wall and physiological and biochemical characters of B34, the fungal was named as Pseudomonas aureofaciens. It was a new antagonistic strain against Thanatephorus cucumeris.

Expression and functional identification of the hypothetical adhesin P32 from Mycoplasma genitalium

Mycoplasma genitalium is the main causative agent for non-gonococcal and non-chlamydial urethritis. P32 is the putative surface-exposed membrane protein of M. genitalium and it has substaintial identity in amino acid sequence with adhesin protein P30 from M. pneumoniae. Since M. pneumoniae mutants lacking P30 protein is defective in cytadherence, P32 protein has been proposed to be an essential adhesin implicated in the adherence of M. genitaliurn to host cells. The prokaryotic expression vector pET-30 （ ＋ ）/p32 was constructed in the present study, and the recombinant protein was expressed in E. coli and purified under denaturing condition. As demonstrated by the immuno- blotting analysis, the recombinant protein could react with rabbit antisera against M. genitalium, and adherence inhibition assays were performed with antisera against this recombinant protein. It was demonstrated that P32 protein apperared to be an adhesion protein of M. genitalium, thus providing the experimental basis for better understanding of the pathogenesis of M. genitalium infection and for the development of the related vaccines against the infection.

MALDI-TOF-TOF鉴定IPG IEF分离的蛋白质

K562细胞是一株分化差、恶性程度高的人白血病细胞.研究表明,在一些分化诱导剂的作用下,细胞可以向红细胞系、粒细胞系和巨K562核细胞系方向分化成熟,并表现出相应血细胞类型的成熟标志.用一维固相pH梯度等电聚焦电泳(LPG IEF)分离K562细胞总蛋白质,其每一条带常包含多个蛋白质,难以用肽指纹谱技术来鉴定.……

Proteomics for discovery of candidate colorectal cancer biomarkers

Colorectal cancer(CRC)is the second most common cause of cancer-related deaths in Europe and other Western countries,mainly due to the lack of wellvalidated clinically useful biomarkers with enough sensitivity and specificity to detect this disease at early stages.Although it is well known that the pathogenesis of CRC is a progressive accumulation of mutations in multiple genes,much less is known at the proteome level.Therefore,in the last years many proteomic studies have been conducted to find new candidate protein biomarkers for diagnosis,prognosis and as therapeutic targets for this malignancy,as well as to elucidate the molecular mechanisms of colorectal carcinogenesis.An important advantage of the proteomic approaches is the capacity to look for multiple differentially expressed proteins in a single study.This review provides an overview of the recent reports describing the different proteomic tools used for the discovery of new protein markers for CRC such as two-dimensional electrophoresis methods,quantitative mass spectrometry-based techniques or protein microarrays.Additionally,we will also focus on the diverse biological samples used for CRC biomarker discovery such as tissue,serum and faeces,besides cell lines and murine models,discussing their advantages and disadvantages,and summarize the most frequently identified candidate CRC markers.

Discovery and identification of Serum Amyloid A protein elevated in lung cancer serum

Two hundred and eighteen serum samples from 175 lung cancer patients and 43 healthy individuals were analyzed by using Surface Enhaced Laser Desorption/Ionization Time of Flight Mass Spectrome- try (SELDI-TOF-MS). The data analyzed by both Biomarker Wizard? and Biomarker Patterns? software showed that a protein peak with the molecular weight of 11.6 kDa significantly increased in lung cancer. Meanwhile,the level of this biomarker was progressively increased with the clinical stages of lung cancer. The candidate biomarker was then obtained from tricine one-dimensional sodium dodecyl sul- fate-polyacrylamide gel electrophoresis by matching the molecular weight with peaks on WCX2 chips and was identified as Serum Amyloid A protein (SAA) by MALDI/MS-MS and database searching. It was further validated in the same serum samples by immunoprecipitation with commercial SAA antibody. To confirm the SAA differential expression in lung cancer patients, the same set of serum samples was measured by ELISA assay. The result showed that at the cutoff point 0.446(OD value)on the Receiver Operating Characteristic (ROC) curve, SAA could better discriminate lung cancer from healthy indi- viduals with sensitivity of 84.1% and specificity of 80%. These findings demonstrated that SAA could be characterized as a biomarker related to pathological stages of lung cancer.

Identification of two new mu-adaptin-related proteins,mu-ARP1 and mu-ARP2

We report the cDNA cloning, primary structure and tissue distribution of two new proteins homologous to mu adaptins, the medium chains of the clathrin coat adaptor complexes. Both predicted proteins share 60% amino acid sequence identity and 27% 31% identity with mu 1 adaptin (ap47) and mu 2 adaptin (ap50). Lower similarity (23% 25% identity) is found with two other mu adaptin related proteins, p47A/B, and there is similarity in the N terminal 150 amino acids Institut fur Physiologische Chemie, Medizinische Fakultat, Ruhr Universitat Bochum, Germany (Wang XL and Kilimann MW) with the adaptin small chains and delta COP. The mRNAs of both molecules are expressed in all tissues analyzed, but with different profiles of relative abundance. mu ARP1 is most abundant in the brain, ovary and lung, whereas mu ARP2 is prominently expressed in the testis. These proteins suggest the existence of uncharacterized types of clathrin or non clathrin associated protein coats in cellular membrane traffic, probably prototype subuits, which provide molecular markers and probes for their characterization.

Analysis of the Arabidopsis Floral Proteome:Detection of over 2000 Proteins and Evidence for Posttranslational Modifications

The proteome of the Arabidopsis flower has not been extensively studied previously. Here, we report a proteomic analysis of the wild type Arabidopsis flower. Using both two-dimensional electrophoresis/mass spectrometry （2-DGEIMS） and multi-dimensional protein identification technology （MudPIT） approaches, we identified 2 446 proteins. Although a single experiment or analysis uncovered only a subset of the proteins we identified, a combination of multiple experiments and analyses facilitated the detection of a greater number of proteins. When proteins are grouped according to RNA expression levels revealed by microarray experiments, we found that proteins encoded by genes with relatively high levels of expression were detected with greater frequencies. On the other hand, at the level of the individual gene/protein, there was not a good correlation between protein spot intensity and microarray values. We also obtained strong evidence for post-translational modification from 2-DGE and MudPIT data. We detected proteins that are annotated to function in protein synthesis, folding, modification, and degradation, as well as the presence of regulatory proteins such as transcription factors and protein kinases. Finally, sequence and evolutionary analysis of genes for active methyl group metabolisms suggests that these genes are highly conserved. Our results allow the formulation of hypotheses regarding post-translational regulation of proteins in the flower, providing new understanding about Arabidopsis flower development and physiology.

A rapid protein sample preparation method based on organic-aqueous microwave irradiation technique

Fast and efficient sample preparation methods are a prerequisite for protein identification in bottom-up proteomics. Here, an innovative microwave irradiation sample preparation method was developed based on an optimized organic-aqueous solvent system for protein identification. Specifically, protein solutions containing high-concentration acetonitrile were subjected to 5 min microwave irradiation. After cooling down, trypsin was added and the digestion was performed with 30 s microwave irradiation, and the resulting peptides were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF MS). A shortened processing time of only 5.5 min is needed with this method(more than 12 h is necessary in the traditional overnight protein sample preparation). Moreover, due to the absence of urea and other chaotropic reagents, the digests can be readily identified by MALDI-TOF MS. When an assessment of this method was performed by digesting a model protein BSA, 69% ± 3% sequence coverage corresponding to 47 ± 3 peptides was obtained, which shows better protein identification than that from the standard overnight protein sample preparation method(51% ± 2% sequence coverage and 23 ± 1 peptides). Another model protein α-casein was used for the analysis of protein phosphorylation with the newly developed method that yielded 4 phosphopeptides with 8 phosphorylation sites, whereas 3 phosphopeptides with 2 phosphorylation sites were obtained from the traditional overnight approach. Moreover, the organic-aqueous microwave irradiation method provides effective digestion for proteins down to fmol.

Application of Proteomics in the Study of Tumor Metastasis

Tumor metastasis is the dominant cause of death in cancer patients. However, the molecular and cellular mechanisms underlying tumor metastasis are still elusive. The identi?cation of protein molecules with their expressions correlated to the metastatic process would help to understand the metastatic mechanisms and thus facilitate the development of strategies for the therapeutic interventions and clini- cal management of cancer. Proteomics is a systematic research approach aiming to provide the global characterization of protein expression and function under given conditions. Proteomic technology has been widely used in biomarker discovery and pathogenetic studies including tumor metastasis. This article provides a brief review of the application of proteomics in identifying molecular factors in tumor metastasis process. The combination of proteomics with other experimental ap- proaches in biochemistry, cell biology, molecular genetics and chemistry, together with the development of new technologies and improvements in existing method- ologies will continue to extend its application in studying cancer metastasis.

A novel organic-inorganic hybrid monolith for trypsin immobilization

In proteomics, attention has focused on various immobilized enzyme reactors (IMERs) for the realization of high throughput digestion. In this report, a novel organic-inorganic hybrid monolith based IMER was prepared in a 100 μm i.d. capillary with 3-glycidoxypropyltrimethoxysilane (GLYMO) as the monomer and tetraethoxysilane (TEOS) as the crosslinker. Trypsin immobilization was achieved via the reaction between vicinal diol groups, which were obtained from hydrolysis of epoxy groups, and the amino groups of trypsin. Bovine serum albumin was digested thoroughly by this IMER in 47 s. After micro-reverse phase liquid chromatography-tandem mass spectrometry (μRPLC-MS/MS) analysis and database searching, beyond 35% sequence coverage was obtained, and the result was comparable to that of 12 h in solution digestion. The present IMER has potential for high throughput digestion.

Sol-gel-derived Poly（dimethylsiloxane） Enzymatic Reactor for Microfluidic Peptide Mapping

The silica-based poly（dimethylsiloxane） （PDMS） microfluidic enzymatic reactor was reported along with its analytical features in coupling with MALDI TOF and ESI MS. Microfluidic chip was fabricated using PDMS casting and O2-plasma techniques, and used for the preparation of enzymatic reactor. Plasma oxidation for PDMS enabled the channel wall of microfluidics to present a layer of silanol （SiOH） groups. These SiOH groups as anchors onto the microchannel wall were linked covalently with the hydroxy groups of trypsin-encapsulated sol matrix. As a result, the leakage of sol-gel matrix from the microchannel was effectively prevented. On-line protein analysis was performed with the microfluidic enzymatic reactor by attachment of stainless steel tubing electrode and replaceable tip, The success of trypsin encapsulation was investigated by capillary electrophoresis （CE） detection, and MALDI TOF and ESI MS analysis. The lab-made device provided excellent extent of digestion even at the fast flow rate of 7.0 μL/min with very short residence time of ca. 2 s. In addition, the encapsulated trypsin exhibits increased stability even after continuous use. These features are the most requisite for high-throughput protein identification.