scholarly journals Glycoinositol phospholipid anchor and protein C-terminus of bovine erythrocyte acetylcholinesterase: analysis by mass spectrometry and by protein and DNA sequencing

1996 ◽  
Vol 314 (3) ◽  
pp. 817-825 ◽  
Author(s):  
Robert HAAS ◽  
Brent C. JACKSON ◽  
Bruce REINHOLD ◽  
John D. FOSTER ◽  
Terrone L. ROSENBERRY
Keyword(s):  
Mass Spectrometry ◽  
Amino Acid ◽  
Dna Sequencing ◽  
Protein Sequencing ◽  
Peptide Sequence ◽  
Partial Reduction ◽  
Bovine Erythrocyte ◽  
Gpi Anchor ◽  
Amino Acid Peptide ◽  
Fragmentation Patterns

Purified bovine erythrocyte acetylcholinesterase (AChE) was radiomethylated on its amine groups and incubated with bacterial phosphatidylinositol-specific phospholipase C to remove the lipid portion of the AChE glycoinositol phospholipid (GPI) anchor, and a C-terminal tryptic fragment that contained the residual GPI glycan was isolated by HPLC. Analysis by electrospray-ionization mass spectrometry revealed a parent ion of m/z 3798. The fragmentation patterns produced by collision-induced dissociation mass spectrometry of the +4 and +5 states of the parent ion indicated a 23-amino acid peptide in amide linkage to ethanolamine-PO4-Hex-Hex-Hex(PO4-ethanolamine) (HexNAc)-HexN(Me)2-inositol phosphate. The glycan structure is completely consistent with that obtained previously for the GPI anchor of human erythrocyte AChE except for the addition of the HexNAc substituent. A nearly complete peptide sequence was deduced from the fragmentation patterns, although four assignments were based only on single fragments of very low abundance. To resolve this uncertainty, a segment of bovine genomic DNA corresponding to the C-terminal AChE sequence was amplified by PCR. DNA sequencing established the 23-amino acid peptide sequence to be FLPKLLSATASEAPCTCSGPAHG, in agreement with the MS data and consistent with results from Edman protein sequencing. Dimerization of AChE polypeptides is mediated by intersubunit disulphide bonding in this C-terminal segment, but the bovine AChE contained two cysteine residues in a …CTC… motif, in contrast with human AChE which contains only a single cysteine in this segment. Although bovine AChE contained no free thiol groups reactive with iodo[14C]acetamide, partial reduction and alkylation with iodo[14C]acetamide revealed that conversion into monomers occurred with an overall incorporation of only one alkyl group per monomer. An identical level of alkylation was observed when dimeric human AChE was converted into monomers by partial reduction. The question of whether the bovine AChE contains one or two intersubunit disulphide linkages is considered.

scholarly journals Real-time dynamic single-molecule protein sequencing on an integrated semiconductor device

2022 ◽  
Author(s):  
Brian D Reed ◽  
Michael J Meyer ◽  
Valentin Abramzon ◽  
Omer Ad ◽  
Pat Adcock ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Dna Sequencing ◽  
Real Time ◽  
Single Molecule ◽  
Dynamic Range ◽  
Protein Sequencing ◽  
Peptide Sequence ◽  
Single Amino Acid ◽  
Functional Components

Proteins are the main structural and functional components of cells, and their dynamic regulation and post-translational modifications (PTMs) underlie cellular phenotypes. Next-generation DNA sequencing technologies have revolutionized our understanding of heredity and gene regulation, but the complex and dynamic states of cells are not fully captured by the genome and transcriptome. Sensitive measurements of the proteome are needed to fully understand biological processes and changes to the proteome that occur in disease states. Studies of the proteome would benefit greatly from methods to directly sequence and digitally quantify proteins and detect PTMs with single-molecule sensitivity and precision. However current methods for studying the proteome lag behind DNA sequencing in throughput, sensitivity, and accessibility due to the complexity and dynamic range of the proteome, the chemical properties of proteins, and the inability to amplify proteins. Here, we demonstrate single-molecule protein sequencing on a compact benchtop instrument using a dynamic sequencing by stepwise degradation approach in which single surface-immobilized peptide molecules are probed in real-time by a mixture of dye-labeled N-terminal amino acid recognizers and simultaneously cleaved by aminopeptidases. By measuring fluorescence intensity, lifetime, and binding kinetics of recognizers on an integrated semiconductor chip we are able to annotate amino acids and identify the peptide sequence. We describe the expansion of the number of recognizable amino acids and demonstrate the kinetic principles that allow individual recognizers to identify multiple amino acids in a highly information-rich manner that is sensitive to adjacent residues. Furthermore, we demonstrate that our method is compatible with both synthetic and natural peptides, and capable of detecting single amino acid changes and PTMs. We anticipate that with further development our protein sequencing method will offer a sensitive, scalable, and accessible platform for studies of the proteome.

scholarly journals Angiogenesis Inhibition by a Short 13 Amino Acid Peptide Sequence of Tetrastatin, the α4(IV) NC1 Domain of Collagen IV

2020 ◽  
Vol 8 ◽  
Author(s):  
Alexia Vautrin-Glabik ◽  
Jérôme Devy ◽  
Camille Bour ◽  
Stéphanie Baud ◽  
Laurence Choulier ◽  
...  
Keyword(s):  
Amino Acid ◽  
Collagen Iv ◽  
Peptide Sequence ◽  
Angiogenesis Inhibition ◽  
Amino Acid Peptide ◽  
Nc1 Domain

scholarly journals Characterization of Cichopeptins, New Phytotoxic Cyclic Lipodepsipeptides Produced by Pseudomonas cichorii SF1-54 and Their Role in Bacterial Midrib Rot Disease of Lettuce

2015 ◽  
Vol 28 (9) ◽  
pp. 1009-1022 ◽  
Author(s):  
Chien-Jui Huang ◽  
Ellen Pauwelyn ◽  
Marc Ongena ◽  
Delphine Debois ◽  
Valerie Leclère ◽  
...  
Keyword(s):  
Amino Acid ◽  
Glycine Betaine ◽  
Sequence Similarity ◽  
Similar Rate ◽  
Peptide Sequence ◽  
Pseudomonas Cichorii ◽  
Cyclic Lipopeptides ◽  
Amino Acid Peptide ◽  
Fatty Acid Chain ◽  
Tomato Pathogen

The lettuce midrib rot pathogen Pseudomonas cichorii SF1-54 produces seven bioactive compounds with biosurfactant properties. Two compounds exhibited necrosis-inducing activity on chicory leaves. The structure of the two phytotoxic compounds, named cichopeptin A and B, was tentatively characterized. They are related cyclic lipopeptides composed of an unsaturated C12-fatty acid chain linked to the N-terminus of a 22–amino acid peptide moiety. Cichopeptin B differs from cichopeptin A only in the last C-terminal amino acid residue, which is probably Val instead of Leu/Ile. Based on peptide sequence similarity, cichopeptins are new cyclic lipopeptides related to corpeptin, produced by the tomato pathogen Pseudomonas corrugata. Production of cichopeptin is stimulated by glycine betaine but not by choline, an upstream precursor of glycine betaine. Furthermore, a gene cluster encoding cichopeptin synthethases, cipABCDEF, is responsible for cichopeptin biosynthesis. A cipA-deletion mutant exhibited significantly less virulence and rotten midribs than the parental strain upon spray inoculation on lettuce. However, the parental and mutant strains multiplied in lettuce leaves at a similar rate. These results demonstrate that cichopeptins contribute to virulence of P. cichorii SF1-54 on lettuce.

Ribosomal proteins in halobacteria

1989 ◽  
Vol 35 (1) ◽  
pp. 195-199 ◽  
Author(s):  
Makoto Kimura ◽  
Evelyn Arndt ◽  
Tomomitsu Hatakeyama ◽  
Tamiko Hatakeyama ◽  
Junko Kimura
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Dna Sequencing ◽  
Glutamic Acid ◽  
Ribosomal Proteins ◽  
Chemical Method ◽  
Amino Acid Sequences ◽  
Protein Sequencing ◽  
Arginine Residues

The amino acid sequences of 16 ribosomal proteins from archaebacterium Halobacterium marismortui have been determined by a direct protein chemical method. In addition, amino acid sequences of three proteins, S11, S18, and L25, have been established by DNA sequencing of their genes as well as by protein sequencing. Comparison of their sequences with those of ribosomal proteins from other organisms revealed that proteins S14, S16, S19, and L25 are related to both eukaryotic and eubacterial ribosomal proteins, being more homologous to eukaryotic than eubacterial counterparts, and proteins S12, S15, and L16 are related to only eukaryotic ribosomal proteins. Furthermore, some proteins are found to be similar to only eubacterial proteins, whereas other proteins show no homology to any other known ribosomal proteins. Comparisons of amino acid compositions between halophilic and nonhalophilic ribosomal proteins revealed that halophilic proteins gain asparatic and glutamic acid residues and significantly lose lysine and arginine residues. In addition, halophilic proteins seem to lose isoleucine as compared with Escherichia coli ribosomal proteins.Key words: halobacteria, ribosomal proteins, amino acid sequence.

Comparison between the Backbone Dynamics of an 11-Amino Acid Peptide Sequence in α-Helical and β-Hairpin Structural Contexts†

Biochemistry ◽  
2006 ◽  
Vol 45 (37) ◽  
pp. 11179-11189 ◽  
Author(s):  
Virginia A. Jarymowycz ◽  
Ewa Krupinska ◽  
Martin J. Stone
Keyword(s):  
Amino Acid ◽  
Backbone Dynamics ◽  
Peptide Sequence ◽  
Amino Acid Peptide

Stimulation of gonadotropin release by a non-GnRH peptide sequence of the GnRH precursor

Science ◽  
1986 ◽  
Vol 232 (4746) ◽  
pp. 68-70 ◽  
Author(s):  
RP Millar ◽  
PJ Wormald ◽  
RC Milton
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Basic Amino Acid ◽  
Prolactin Secretion ◽  
Biologically Active ◽  
Peptide Sequence ◽  
Pituitary Cells ◽  
Amino Acid Residues ◽  
Amino Acid Peptide ◽  
Gonadotropin Release

The human gonadotropin-releasing hormone (GnRH) precursor comprises the GnRH sequence followed by an extension of 59 amino acids. Basic amino acid residues in the carboxyl terminal extension may represent sites of processing to biologically active peptides. A synthetic peptide comprising the first 13 amino acids (H X Asp-Ala-Glu-Asn-Leu-Ile-Asp-Ser-Phe-Gln-Glu-Ile-Val X OH) of the 59-amino acid peptide was found to stimulate the release of gonadotropic hormones from human and baboon anterior pituitary cells in culture. The peptide did not affect thyrotropin or prolactin secretion. A GnRH antagonist did not inhibit gonadotropin stimulation by the peptide, and the peptide did not compete with GnRH for GnRH pituitary receptors, indicating that the action of the peptide is independent of the GnRH receptor. The GnRH precursor contains two distinct peptide sequences capable of stimulating gonadotropin release from human and baboon pituitary cells.

scholarly journals Post-translational Modifications of Arabinogalactan-peptides ofArabidopsis thaliana

2004 ◽  
Vol 279 (44) ◽  
pp. 45503-45511 ◽  
Author(s):  
Carolyn J. Schultz ◽  
Kris L. Ferguson ◽  
Jelle Lahnstein ◽  
Antony Bacic
Keyword(s):  
Mass Spectrometry ◽  
Experimental Evidence ◽  
Cleavage Site ◽  
Expressed Sequence Tag ◽  
Protein Sequencing ◽  
Plant Proteins ◽  
Gpi Anchor ◽  
Post Translational Modifications

We have developed a method for separating the deglycosylated protein/peptide backbones of the small arabinogalactan (AG)-peptides from the larger classical arabinogalactan-proteins (AGPs). AGPs are an important class of plant proteoglycans implicated in plant growth and development. Separation of AG-peptides enabled us to identify eight of 12 AG-peptides fromArabidopsis thalianapredicted from genomic sequences. Of the remaining four, two have low abundance based on expressed sequence tag databases and the other two are only present in pollen (At3g20865) or flowers (At3g57690) and therefore would not be detected in our analysis. Characterization of AG-peptides was performed using matrix-assisted laser desorption ionization-time of flight mass spectrometry and tandem mass spectrometry protein sequencing. These data provide (i) experimental evidence that AG-peptides are processedin vivofor the addition of a glycosylphosphatidylinositol (GPI) anchor, (ii) cleavage site information for both the endoplasmic reticulum secretion signal and the GPI-anchor signal for eight of the 12 AG-peptides, and (iii) experimental evidence that the Gly-Pro motif is hydroxylatedin vivo. Furthermore, we show that AtAGP16 is GPI-anchored despite its unusually long hydrophobic C-terminal GPI-signal sequence. Prior to this work, the GPI-anchor cleavage site for only two plant proteins, NaAGP1 fromNicotiana alataand PcAGP1 fromPyrus communis, had been determined experimentally. Characterization of the post-translational modifications of AG-peptides contributes toward obtaining the complete primary structure of this class of biologically important plant proteoglycans and provides a greater understanding of post-translational modifications of plant proteins.

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