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Nucleic acid associated proteins (NAaP) play the essential roles in gene regulation and protein expression. The global analysis of cellular NAaP would give a broad insight to understand the interaction between nucleic acids and the associated proteins, such as the important proteinous regulation factors on nucleic acids. Proteomic analysis presents a novel strategy to investigate a group of proteins. However, the large scale analysis of NAaP is yet impossible due to the lack of approaches to harvest target protein groups with a high efficiency. Herein, a simple and efficient method was developed to collect cellular NAaP using magnetic oxidized carbon nanotubes based on the strong interaction between carbon nanotubes and nucleic acids along with corresponding associated proteins. We found that the magnetic oxidized carbon nanotubes demonstrated a nearly 100% extraction efficiency for intracellular nucleic acids from cells in vitro. Importantly, the proteins associated on nucleic acids could be highly efficiently harvested using magnetic oxidized carbon nanotubes due to the binding of NAaP on nucleic acids. 1594 groups of nuclear NAaP and 2595 groups of cellular NAaP were extracted and identified from about 1,000,000 cells, and 803 groups of NAaP were analyzed with only about 10,000 cells, showing a promising performance for the proteomic analysis of NAaP from minute cellular samples. This highly efficient extraction strategy for NAaP is a simple approach to identify cellular nucleic acid associated proteome, and we believed this strategy could be further applied in systems biology to understand the gene expression and regulation.

Various enzyme reactors and online enzyme digestion strategies have been developed in recent years. These reactors greatly enhanced the detection sensitivity and proteome coverage in qualitative proteomics. However, these devices have higher rates of miscleavage in protein digestion. Therefore, we investigated the effect of online enzyme digestion on the quantification accuracy of quantitative proteomics using chemical or metabolic isotope labeling approaches. The incomplete digestion would introduce some unexpected variations in comparative quantification when the samples are digested and then chemically isotope labeled in different aliquots. Even when identical protein aliquots are processed on these devices using post-digestion chemical isotope labeling and the CVs of the ratios controlled to less than 50% in replicate analyses, about 10% of the quantified proteins have a ratio greater than two-fold, whereas in theory the ratio is 1:1. Interestingly, the incomplete digestion with enzyme reactor is not a problem when metabolic isotope labeling samples were processed because the proteins are isotopically labeled in vivo prior to their simultaneous digestion within the reactor. Our results also demonstrated that both high quantification accuracy and high proteome coverage can be achieved in comparative proteome quantification using online enzyme digestion even when a limited amount of metabolic isotope labeling samples is used (1683 proteins comparatively quantified from 10(5) Hela cells).

Complete coverage of all phosphorylation sites in a proteome is the ultimate goal for large-scale phosphoproteome analysis. However, only making use of one protease trypsin for protein digestion cannot cover all phosphorylation sites, because not all tryptic phosphopeptides are detectable in MS. To further increase the phosphoproteomics coverage of HeLa cells, we proposed a tandem digestion approach by using two different proteases. By combining the data set of the first Glu-C digestion and the second trypsin digestion, the tandem digestion approach resulted in the identification of 8062 unique phosphopeptides and 8507 phosphorylation sites in HeLa cells. The conventional trypsin digestion approach resulted in the identification of 3891 unique phosphopeptides and 4647 phosphorylation sites. It was found that the phosphorylation sites identified from the above two approaches were highly complementary. By combining above two data sets, in total we identified 10899 unique phosphopeptides and 11262 phosphorylation sites, corresponding to 3437 unique phosphoproteins with FDR < 1% at peptide level. We also compared the kinase motifs extracted from trypsin, Glu-C, or a second trypsin digestion data sets. It was observed that basophilic motifs were more frequently found in the trypsin and the second trypsin digestion data sets, and the acidic motifs were more frequently found in the Glu-C digestion data set. These results demonstrated that our tandem digestion approach is a good complement to the conventional trypsin digestion approach for improving the phosphoproteomics analysis coverage of HeLa cells.

Deciphering the kinase-substrate relationship is vital for the study of phosphorylation network. The use of immobilized proteins on protein chip as the library for screening of potential kinase substrates is a tried-and-tested method. However, information on phosphorylation sites is lacking and the creation of the library with proteins of whole proteome by recombinant expression is costly and difficult. In this study, a new solid-phase approach by immobilization of proteins from cell lysate onto beads as a protein library for kinase substrate screening was developed. It was found that consensus phosphorylation sites motif for kinase substrates could be accurately determined and hundreds of in vitro kinase substrates and their phosphorylation sites could be identified by using this method.

A line of double-transgenic mice that develop neoplasms arising primarily in the pancreas was established. In these mice, the oncogene SV40 T antigen (Tag) was detected in the pancreas with and without the control of Tet-on system. The transgenic mice that developed pancreatic tumors as early as 20 weeks of age showed hypoglycemia on a blood glucose test. Pathological and immunohistochemical characterizations demonstrated that the tumors belonged to neuroendocrine neoplasms arising from pancreatic islets. A change in IGFs/IGF-1R signaling pathway was detected using real-time PCR analysis. A potential association between the IGFs/IGF-1R system and SV40Tag was studied to further explain the cancerogenesis of the double-transgenic mice by Western blot analysis and immunoprecipitation experiments. The results suggest that a Tag transgenic mice model could be used to study the molecular mechanism of the tumorigenesis of islets.

MS1 full scan based quantification is one of the most popular approaches for large-scale proteome quantification. Typically only three different samples can be differentially labeled and quantified in a single experiment. Here we present a two stages stable isotope labeling strategy which allows six different protein samples (six-plex) to be reliably labeled and simultaneously quantified at MS1 level. Briefly in the first stage, isotope lysine-d0 (K0) and lysine-d4 (K4) are in vivo incorporated into different protein samples during cell culture. Then in the second stage, three of K0 and K4 labeled protein samples are digested by lysine C and in vitro labeled with light (2CH3), medium (2CD2H), and heavy (2(13)CD3) dimethyl groups, respectively. We demonstrated that this six-plex isotope labeling strategy could successfully investigate the dynamics of protein turnover in a high throughput manner.

Substrate motif is of great importance in kinase-substrate recognition and contributes a lot to the understanding of signal transduction. In this study, a proteomic peptide library was generated to determine substrate motif of casein kinase 2. Firstly, whole cell lysate was digested by trypsin to generate a large number of candidate peptides, which were then incubated with alkaline phosphotase to dephosphorylate intrinsic phosphopeptides. Then, the unphosphorylated peptide mixture was incubated with casein kinase 2 (CK2) and adenosine-triphosphate (ATP) for 30 min, and the resulting phosphopeptides were enriched by immobilized metal affinity chromatography (IMAC) followed by reversed-phase liquid chromatographic separation coupled with tandem mass spectrometry detection. Finally, the 472 unique phosphopeptides and 451 unique phosphorylation sites were identified, resulting in the determination of a motif of S/T-D/E-x-D/E for CK2. This method enables accurate determination of substrate motif in a short time and can be readily applied to other kinases.

A transgenic mouse model expressing Simian virus 40 T-antigen (SV40Tag) under the control of a tetracycline system was generated. In this model, a cerebellar tumor was developed after doxycycline hydrochloride treatment. Real time-polymerase chain reaction and immunohistochemistry results indicated that the SV40Tag gene was expressed in the tumor. Pathological analysis showed that the tumor belonged to medulloblastoma. Further molecular characterization of the tumor demonstrated that the insulin-like growth factor (IGF) signaling pathway was activated. We also found that the SV40Tag could bind and translocate insulin receptor substrate 1 into the nucleus in primary cultured tumor cells. The interaction between the IGF pathway and SV40Tag may contribute to the process of malignant transformation in medulloblastoma. This transgenic animal model provides an important tool for studies on the signal pathways involved in the preneoplastic process in medulloblastoma and could help to identify therapeutic targets for brain tumors.

AIM: To generate a SV40Tag transgenic tumor animal model and to study the mechanism underlying tumorigenesis. METHODS: A mammary gland expression vector containing SV40Tag DNA was generated. Transgene fragments were microinjeted into fertilized eggs of FVB mice. The genetically manipulated embryos were transferred into the oviducts of pseudo-pregnant female mice. PCR and Northern blot analysis were used for genotype analysis of F1 and F2 mice. Transgene expression was detected by RT-PCR and immunohistochemistry. RESULTS: SV40Tag gene was detected in two lines of transgenic mice. One of them delivered the transgene to F1 and a tumor was found in the pancreas of these mice. RT-PCR and immunohistochemistry showed that SV40Tag gene was expressed in the tumor. Pathological characterization of the transgenic mice demonstrated that the tumor belonged to pancreatic cystic neoplasm. CONCLUSION: SV40Tag transgenic mouse model can be successfully established. The transgenic mice develop a pancreatic tumor, which can be used for investigation of the molecular mechanism of tumorigenesis in vivo.

AIM: To study the gene expression changes in pancreatic cystic neoplasm in SV40Tag transgenic mice model and to provide information about the prevention, clinical diagnosis and therapy of pancreatic cancer. METHODS: Using the pBC-SV40Tag transgenic mice model of pancreatic cystic neoplasm, we studied the gene expression changes by applying high-density microarrays. Validation of part gene expression profiling data was performed using real-time PCR. RESULTS: By using high-density oligonucleotide microarray, of 14 113 genes, 453 were increased and 760 decreased in pancreatic cystic neoplasm, including oncogenes, cell-cycle-related genes, signal transduction-related genes, skeleton-related genes and metabolism-related genes. Among these, we confirmed the changes in Igf, Shh and Wnt signal pathways with real-time PCR. The results of real-time PCR showed similar expression changes in gene chip. CONCLUSION: all the altered expression genes are associated with cell cycle, DNA damage and repair, signal pathway, and metabolism. SV40Tag may cooperate with several proteins in promoting tumorigenesis.