Identification of foetal brain proteins by two‐dimensional gel electrophoresis and mass spectrometry

Oppermann, Madalina; Cols, Neus; Nyman, Tuula A.; Helin, Jari; Saarinen, Jyrki; Byman, Irene; Torán, Núria; Alaiya, Ayodele; Bergman, Tomas; Kalkkinen, Nisse; Gonzàlez‐Duarte, Roser; Jörnvall, Hans

doi:10.1046/j.1432-1327.2000.01524.x

Cited by 34 publications

(30 citation statements)

References 27 publications

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“…The stained protein band was cut from the gel and placed in an Eppendorf tube for in-gel digestion (25). Washing was carried out in 0.2 M ammonium bicarbonate containing 50% acetonitrile.…”

Section: Methodsmentioning

confidence: 99%

Identification of Human Intestinal Alkaline Sphingomyelinase as a Novel Ecto-enzyme Related to the Nucleotide Phosphodiesterase Family

Duan

Bergman

et al. 2003

Journal of Biological Chemistry

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129

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Alkaline sphingomyelinase (alk-SMase) hydrolyzes dietary sphingomyelin and generates sphingolipid messengers in the gut. In the present study, we purified the enzyme, identified a part of the amino acid sequence, and found a cDNA in the GenBank TM coding for the protein. The cDNA contains 1841 bp, and the open reading frame encodes 458 amino acids. Transient expression of the cDNA linked to a Myc tag in COS-7 cells increased alk-SMase activity in the cell extract by 689-fold and in the medium by 27-fold. High activity was also identified in the anti-Myc immunoprecipitated proteins and the proteins cross-reacted with anti-human alkSMase. Northern blotting of human intestinal tissues found high levels of alk-SMase mRNA in the intestine and liver. The amino acid sequence shared no similarity with acid and neutral SMases but was related to the ecto-nucleotide phosphodiesterase (NPP) family with 30 -36% identity to human NPPs. Alk-SMase has a predicted signal peptide domain at the N terminus and a signal anchor domain at the C terminus. The ion-binding sites and the catalytic residue of NPPs were conserved, but the substrate specificity domain was modified. Alk-SMase had no detectable nucleotidase activity, but its activity against sphingomyelin could be inhibited by orthovanadate, imidazole, and ATP. In contrast to NPPs, alk-SMase activity was not stimulated by divalent metal ions but inhibited by Zn 2؉ . Differing from NPP2, the alk-SMase cleaved phosphocholine but not choline from lysophosphatidylcholine. Phylogenetic tree indicated that the enzyme is a new branch derived from the NPP family. Two cDNA sequences of mouse and rat that shared 83% identity to human alk-SMase were identified in the GenBank TM . In conclusion, we identified the amino acid and cDNA sequences of human intestinal alk-SMase, and found that it is a novel ectoenzyme related to the NPP family with specific features essential for its SMase activity. Sphingomyelin (SM)1 is a component of all mammalian cell membranes particularly the plasma membrane and the lysosomal membrane. SM is also a dietary component and is mainly present in milk, eggs, meat, and marine products (1, 2). Hydrolysis of SM generates ceramide, sphingosine, and sphingosine 1-phosphate that have regulatory effects on numerous cellular functions such as proliferation, differentiation, and apoptosis (3, 4). At least five types of sphingomyelinase (SMase) have been identified, of which acid and neutral SMases have been cloned (5-9). An enzyme that catalyzes hydrolysis of SM with optimal alkaline pH was first identified in the intestinal content of human and intestinal mucosa of rat and pig by Nilsson (10) and was named alkaline SMase (alkSMase) thereafter (11). Previous studies indicated that alkSMase may be responsible for digestion of dietary SM and for hydrolysis of endogenous SM derived from bile and from the brush borders of sloughed mucosal cells.SM metabolism in the intestine may have implications in colon cancer development. Dietary supplement with SM and ceramide analogues...

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“…The stained protein band was cut from the gel and placed in an Eppendorf tube for in-gel digestion (25). Washing was carried out in 0.2 M ammonium bicarbonate containing 50% acetonitrile.…”

Section: Methodsmentioning

confidence: 99%

Identification of Human Intestinal Alkaline Sphingomyelinase as a Novel Ecto-enzyme Related to the Nucleotide Phosphodiesterase Family

Duan

Bergman

et al. 2003

Journal of Biological Chemistry

Self Cite

129

186

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“…The quantity of mRNA in a single cell is Claudio et al 1998;Trendelenburg et al 2002;Bosch et al 2000;Carulli, et al 1998;Oppermann et al 2000. # Some journals used as protein concentration approximately 1.0 pg (about 5 x 10 5 molecules) (Ambion, 2004).…”

Section: Single-cell Samplingmentioning

confidence: 99%

A strategy to identify genomic expression at single-cell level or a small number of cells

2005

Electro Journal of Biotech

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Recent advances in functional genomics allow us to estimate the expression of several thousands of genes in the mammalian genome. Techniques such as microarrays, expressed tag sequencing (EST), serial analysis of gene expression (SAGE), subtractive cloning and differential display (DD), and two-dimensional electrophoresis gel have been extensively used to screen and analyze parallel gene expression. Some pathological processes, for example, tumorigenesis and solid tumour growth, in which the former is derived from a single-cell and the latter has a mixed-cell problem, present new challenges to the limit of these functional genomic techniques. To fully understand the functions of cells in tumorigenesis or in heterogeneous solid tumour masses, it is essential for scientists and physicians to develop a strategy to identify genomic expression profiles for a single-cell or for small numbers of cells. In this article, we review recently developed methods that enable functional genomic analysis at the single-cell or multicell level. In addition, the paper will review different techniques of single-cell genomic expression at the level of DNA, mRNA, protein and post-translational modifications.

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“…Similarly, proteomic techniques have rapidly evolved and have the potential to be used to monitor disease specific alterations (Misek et al, 2004). For instance, two-dimensional gel electrophoresis (2DE) can be used to identify differentially expressed peptides and subsequent mass spectrometry can then be used for protein identification (Oppermann et al, 2000). Such approaches enable discernment of tumor specific quantitative or qualitative changes of protein patterns.…”

Section: Introductionmentioning

confidence: 99%

Stage‐specific alterations of the genome, transcriptome, and proteome during colorectal carcinogenesis

Habermann

Paulsen

Roblick

et al. 2006

Genes Chromosomes & Cancer

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To identify sequential alterations of the genome, transcriptome, and proteome during colorectal cancer progression, we have analyzed tissue samples from 36 patients, including the complete mucosa-adenoma-carcinoma sequence from 8 patients. Comparative genomic hybridization (CGH) revealed patterns of stage specific, recurrent genomic imbalances. Gene expression analysis on 9K cDNA arrays identified 58 genes differentially expressed between normal mucosa and adenoma, 116 genes between adenoma and carcinoma, and 158 genes between primary carcinoma and liver metastasis (P < 0.001). Parallel analysis of our samples by CGH and expression profiling revealed a direct correlation of chromosomal copy number changes with chromosome-specific average gene expression levels. Protein expression was analyzed by two-dimensional gel electrophoresis and subsequent mass spectrometry. Although there was no direct match of differentially expressed proteins and genes, the majority of them belonged to identical pathways or networks. In conclusion, increasing genomic instability and a recurrent pattern of chromosomal imbalances as well as specific gene and protein expression changes correlate with distinct stages of colorectal cancer progression. Chromosomal aneuploidies directly affect average resident gene expression levels, thereby contributing to a massive deregulation of the cellular transcriptome. The identification of novel genes and proteins might deliver molecular targets for diagnostic and therapeutic interventions.

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Identification of foetal brain proteins by two‐dimensional gel electrophoresis and mass spectrometry

Cited by 34 publications

References 27 publications

Identification of Human Intestinal Alkaline Sphingomyelinase as a Novel Ecto-enzyme Related to the Nucleotide Phosphodiesterase Family

Identification of Human Intestinal Alkaline Sphingomyelinase as a Novel Ecto-enzyme Related to the Nucleotide Phosphodiesterase Family

A strategy to identify genomic expression at single-cell level or a small number of cells

Stage‐specific alterations of the genome, transcriptome, and proteome during colorectal carcinogenesis

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