Abstract of related literatures |
1. Human alpha 2-HS-glycoprotein (AHSG) is a plasma protein synthesized in liver and selectively concentrated in bone matrix. It has been reported to be involved in bone formation and resorption as well as immune responses. Recently, AHSG was found to be the species equivalent protein of fetuin, the major fetal serum protein in cattle and sheep. The function and regulation of AHSG/fetuin in different species are not understood. We have isolated a liver cDNA clone that encodes the human AHSG/bovine fetuin homologue in the mouse. The AHSG/fetuin gene may have a role in differentiation since it is expressed in mouse limb buds and brain only at certain stages during development. Mouse liver AHSG/fetuin mRNA was present at low level at 12 days gestation but its level increased during the late part of gestation and peaked between 1 to 3 months after birth. The regulation of mouse AHSG/fetuin synthesis during development was found to be significantly different from that of sheep and bovine fetuin. Compared to fetuin, which is reduced in adult to 1 to 2% of the fetal level, mouse AHSG synthesis subsides only 50% 4 months after birth. PMID: [1373325]
2. We proposed that the alpha2-Heremans Schmid glycoprotein/fetuin family of serum proteins inhibits unwanted mineralization. To test this hypothesis in animals, we cloned the mouse fetuin gene and generated mice lacking fetuin. The gene consists of seven exons and six introns. The cystatin-like domains D1 and D2 of mouse fetuin are encoded by three exons each, whereas a single terminal exon encodes the carboxyl-terminal domain D3. The promoter structure is well conserved between rat and mouse fetuin genes within the regions shown to bind transcription factors in the rat system. Expression studies demonstrated that mice homozygous for the gene deletion lacked fetuin protein and that mice heterozygous for the null mutation produced roughly half the amount of fetuin protein produced by wild-type mice. Fetuin-deficient mice were fertile and showed no gross anatomical abnormalities. However, the serum inhibition of apatite formation was compromised in these mice as well as in heterozygotes. In addition, some homozygous fetuin-deficient female ex-breeders developed ectopic microcalcifications in soft tissues. These results corroborate a role for fetuin in serum calcium homeostasis. The fact that generalized ectopic calcification did not occur in fetuin-deficient mice proves that additional inhibitors of phase separation exist in serum. PMID: [9395485]
3. The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5'-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline. PMID: [15489334]
4. A novel trypsin inhibitor, tentatively named countertrypin, was isolated from mouse plasma in an apparently homogeneous state. Countertrypin is a 53-kDa glycoprotein having about 30% carbohydrate, and did not cross-react immunologically with either mouse alpha 1-antiproteinase (also called alpha 1-proteinase inhibitor or alpha 1-antitrypsin) or contrapsin. Countertrypin had no inhibitory activity against chymotrypsin, pancreatic elastase, neutrophil elastase, thrombin, plasmin, plasma kallikrein, pancreatic kallikrein, clotting factor Xa, or papain. This inhibitory spectrum does not correspond to any of the known plasma proteinase inhibitors that have been well characterized in human or other mammals. NH2-terminal amino acid sequence analysis of the intact molecule and three peptides obtained by CNBr digestion revealed that a total of 93 amino acid residues could be aligned with stretches in human alpha 2-HS glycoprotein, bovine fetuin, and rat pp63 (rat fetuin). Human alpha 2-HS glycoprotein and bovine fetuin prepared without use of ethanol inhibited trypsin and pancreatic and neutrophil elastases. These results indicate that mouse countertrypin is a new member of the mammalian fetuin family, which possibly has the trypsin-inhibiting activity in common. PMID: [7688730]
5. A procedure to map N-glycosylation sites is presented here. It can be applied to purified proteins as well as to highly complex mixtures. The method exploits deglycosylation by PNGase F in a diagonal, reverse-phase chromatographic setup. When applied to 10 microL of mouse serum, affinity-depleted for its three most abundant components, 117 known or predicted sites were mapped in addition to 10 novel sites. Several sites were detected on soluble membrane or receptor components. Our method furthermore senses the nature of glycan structures and can detect differential glycosylation on a given site. These properties--high sensitivity and dependence on glycan imprinting--can be exploited for glycan-biomarker analysis. PMID: [16944957]
6. A comprehensive understanding of the mouse plasma proteome is important for studies using mouse models to identify protein markers of human disease. To enhance our analysis of the mouse plasma proteome, we have developed a method for isolating low-abundance proteins using a cysteine-containing glycopeptide strategy. This method involves two orthogonal affinity capture steps. First, glycoproteins are coupled to an azlactone copolymer gel using hydrazide chemistry and cysteine residues are then biotinylated. After trypsinization and extensive washing, tethered N-glycosylated tryptic peptides are released from the gel using PNGase F. Biotinylated cysteinyl-containing glycopeptides are then affinity selected using a monomeric avidin gel and analyzed by LC-MS/MS. We have applied the method to a proteome analysis of mouse plasma. In two independent analyses using 200 muL each of C57BL mouse plasma, 51 proteins were detected. Only 42 proteins were seen when the same plasma sample was analyzed by glycopeptides only. A total of 104 N-glycosylation sites were identified. Of these, 17 sites have hitherto not been annotated in the Swiss-Prot database whereas 48 were considered probable, potential, or by similarity - i.e., based on little or no experimental evidence. We show that analysis by cysteine-containing glycopeptides allows detection of low-abundance proteins such as the epidermal growth factor receptor, the Vitamin K-dependent protein Z, the hepatocyte growth factor activator, and the lymphatic endothelium-specific hyaluronan receptor as these proteins were not detected in the glycopeptide control analysis. PMID: [17330941]
7. Protein phosphorylation is a complex network of signaling and regulatory events that affects virtually every cellular process. Our understanding of the nature of this network as a whole remains limited, largely because of an array of technical challenges in the isolation and high-throughput sequencing of phosphorylated species. In the present work, we demonstrate that a combination of tandem phosphopeptide enrichment methods, high performance MS, and optimized database search/data filtering strategies is a powerful tool for surveying the phosphoproteome. Using our integrated analytical platform, we report the identification of 5,635 nonredundant phosphorylation sites from 2,328 proteins from mouse liver. From this list of sites, we extracted both novel and known motifs for specific Ser/Thr kinases including a "dipolar" motif. We also found that C-terminal phosphorylation was more frequent than at any other location and that the distribution of potential kinases for these sites was unique. Finally, we identified double phosphorylation motifs that may be involved in ordered phosphorylation. PMID: [17242355]
8. The elucidation of protein post-translational modifications, such as phosphorylation, remains a challenging analytical task for proteomic studies. Since many of the proteins targeted for phosphorylation are low in abundance and phosphorylation is typically substoichiometric, a prerequisite for their identification is the specific enrichment of phosphopeptide prior to mass spectrometric analysis. Here, we presented a new method termed as immobilized titanium ion affinity chromatography (Ti (4+)-IMAC) for enriching phosphopeptides. A phosphate polymer, which was prepared by direct polymerization of monomers containing phosphate groups, was applied to immobilize Ti (4+) through the chelating interaction between phosphate groups on the polymer and Ti (4+). The resulting Ti (4+)-IMAC resin specifically isolates phosphopeptides from a digest mixture of standard phosphoproteins and nonphosphoprotein (BSA) in a ratio as low as 1:500. Ti (4+)-IMAC was further applied for phosphoproteome analysis of mouse liver. We also compared Ti (4+)-IMAC to other enrichment methods including Fe (3+)-IMAC, Zr (4+)-IMAC, TiO 2 and ZrO 2, and demonstrate superior selectivity and efficiency of Ti (4+)-IMAC for the isolation and enrichment of phosphopeptides. The high specificity and efficiency of phosphopeptide enrichment by Ti (4+)-IMAC mainly resulted from the flexibility of immobilized titanium ion with spacer arm linked to polymer beads as well as the specific interaction between immobilized titanium ion and phosphate group on phosphopeptides. PMID: [18630941]
9. Kinases play a prominent role in tumor development, pointing to the presence of specific phosphorylation patterns in tumor tissues. Here, we investigate whether recently developed high resolution mass spectrometric (MS) methods for proteome and phosphoproteome analysis can also be applied to solid tumors. As tumor model, we used TG3 mutant mice carrying skin melanomas. At total of 100 microg of solid tumor lysate yielded a melanoma proteome of 4443 identified proteins, including at least 88 putative melanoma markers previously found by cDNA microarray technology. Analysis of 2 mg of lysate from dissected melanoma with titansphere chromatography and 8 mg with strong cation exchange together resulted in the identification of more than 5600 phosphorylation sites on 2250 proteins. The phosphoproteome included many hits from pathways important in melanoma. One-month storage at -80 degrees C did not significantly decrease the number of identified phosphorylation sites. Thus, solid tumor can be analyzed by MS-based proteomics with similar efficiency as cell culture models and in amounts compatible with biopsies. PMID: [19367708]
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