scholarly journals The Development of Cleveland Peptide Mapping by Don W. Cleveland

2006 ◽  
Vol 281 (33) ◽  
pp. e27-e28
Author(s):  
Nicole Kresge ◽  
Robert D. Simoni ◽  
Robert L. Hill
Keyword(s):  
Peptide Mapping

scholarly journals Deamidation and glycation of a Bacillus licheniformis α-amylase during industrial fermentation can improve detergent wash performance

Amylase ◽  
2021 ◽  
Vol 5 (1) ◽  
pp. 38-49
Author(s):  
Connie Pontoppidan ◽  
Svend G. Kaasgaard ◽  
Carsten P. Sønksen ◽  
Carsten Andersen ◽  
Birte Svensson
Keyword(s):  
Bacillus Licheniformis ◽  
Mutational Analysis ◽  
Peptide Mapping ◽  
Substrate Binding ◽  
Carbohydrate Binding ◽  
Surface Binding ◽  
Lysine Residues ◽  
Tandem Mass Spectrometric ◽  
Binding Cleft ◽  
Household Detergents

Abstract The industrial thermostable Bacillus licheniformis α-amylase (BLA) has wide applications, including in household detergents, and efforts to improve its performance are continuously ongoing. BLA during the industrial production is deamidated and glycated resulting in multiple forms with different isoelectric points. Forty modified positions were identified by tandem mass spectrometric peptide mapping of BLA forms separated by isoelectric focusing. These modified 12 asparagine, 9 glutamine, 8 arginine and 11 lysine residues are mostly situated on the enzyme surface and several belong to regions involved in stability, activity and carbohydrate binding. Eight residues presumed to interact with starch at the active site and surface binding sites (SBSs) were subjected to mutational analysis. Five mutants mimicking deamidation (N→D, Q→E) at the substrate binding cleft showed moderate to no effect on thermostability and k cat and K M for maltoheptaose and amylose. Notably, the mutations improved laundry wash efficiency in detergents at pH 8.5 and 10.0. Replacing three reducing sugar reactive side chains (K→M, R→L) at a distant substrate binding region and two SBSs enhanced wash performance especially in liquid detergent at pH 8.5, slightly improved enzymatic activity and maintained thermostability. Wash performance was most improved (5-fold) for the N265D mutant near substrate binding subsite +3.

scholarly journals Characterization of a novel + 70 Da modification in rhGM-CSF expressed in E. coli using chemical assays in combination with mass spectrometry

Amino Acids ◽  
2021 ◽  
Author(s):  
Magdalena Widgren Sandberg ◽  
Jakob Bunkenborg ◽  
Stine Thyssen ◽  
Martin Villadsen ◽  
Thomas Kofoed
Keyword(s):  
Mass Spectrometry ◽  
Tandem Mass Spectrometry ◽  
Peptide Mapping ◽  
Covalent Modification ◽  
Peptide Fragmentation ◽  
Tandem Mass ◽  
E Coli ◽  
Gm Csf ◽  
Fragmentation Behavior ◽  
Macrophage Colony

AbstractGranulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine and a white blood cell growth factor that has found usage as a therapeutic protein. During analysis of different fermentation batches of GM-CSF recombinantly expressed in E. coli, a covalent modification was identified on the protein by intact mass spectrometry. The modification gave a mass shift of + 70 Da and peptide mapping analysis demonstrated that it located to the protein N-terminus and lysine side chains. The chemical composition of C4H6O was found to be the best candidate by peptide fragmentation using tandem mass spectrometry. The modification likely contains a carbonyl group, since the mass of the modification increased by 2 Da by reduction with borane pyridine complex and it reacted with 2,4-dinitrophenylhydrazine. On the basis of chemical and tandem mass spectrometry fragmentation behavior, the modification could be attributed to crotonaldehyde, a reactive compound formed during lipid peroxidation. A low recorded oxygen pressure in the reactor during protein expression could be linked to the formation of this compound. This study shows the importance of maintaining full control over all reaction parameters during recombinant protein production.

scholarly journals Design of epidermal growth factor immobilization on 3D biocompatible scaffolds to promote tissue repair and regeneration

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Teodora Bavaro ◽  
Sara Tengattini ◽  
Refaya Rezwan ◽  
Enrica Chiesa ◽  
Caterina Temporini ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Tissue Repair ◽  
Peptide Mapping ◽  
Rnase A ◽  
Fibroblast Cells ◽  
Native State ◽  
Tissue Repair And Regeneration ◽  
Epidermal Growth ◽  
Cell Proliferative Activity

AbstractExogenous application of human epidermal growth factor (hEGF) stimulates epidermal wound healing. The aim of this study was to develop bioconjugates based on hEGF mimicking the protein in its native state and thus suitable for tissue engineering applications, in particular for treating skin-related disorders as burns. Ribonuclease A (RNase A) was used to investigate a number of different activated-agarose carriers: cyanogen bromide (CNBr)-activated-agarose and glyoxyl-agarose showed to preserve the appropriate orientation of the protein for receptor binding. EGF was immobilized on these carriers and immobilization yield was evaluated (100% and 12%, respectively). A peptide mapping of unbound protein regions was carried out by LC–MS to take evidence of the residues involved in the immobilization and, consequently, the flexibility and surface accessibility of immobilized EGF. To assess cell proliferative activities, 10, 25, 50, and 100 ng/mL of each immobilized EGF sample were seeded on fibroblast cells and incubated for 24, 48 and 72 h. The immobilized growth factor showed significantly high cell proliferative activity at 50 and 100 ng/mL compared to control and soluble EGF. Although both of the immobilized samples show dose-dependency when seeded with high number of fibroblast cells, CNBr-agarose-EGF showed a significantly high activity at 100 ng/mL and 72 h incubation, compared to glyoxyl-agarose-EGF.

scholarly journals Studies on the autophosphorylation of the insulin receptor from human placenta. Analysis of the sites phosphorylated by two-dimensional peptide mapping

1988 ◽  
Vol 252 (2) ◽  
pp. 607-615 ◽  
Author(s):  
J M Tavaré ◽  
R M Denton
Keyword(s):  
Thin Layer ◽  
Insulin Receptor ◽  
Peptide Mapping ◽  
Beta Subunit ◽  
Two Dimensional ◽  
Intact Cells ◽  
Tyrosine Residues ◽  
Domain 3 ◽  
Receptor Beta

1. A partially purified preparation of human placental insulin receptors was incubated with [gamma-32P]ATP in the presence or absence of insulin. The 32P-labelled insulin-receptor beta-subunits were then isolated, cleaved with trypsin followed by protease V8 and the [32P]phosphopeptides generated were analysed by thin layer electrophoresis and chromatography. This approach revealed that insulin stimulates autophosphorylation of the insulin-receptor beta-subunit in vitro on at least seven tyrosine residues distributed among three distinct domains. 2. One domain (domain 2), containing tyrosine residues 1146, 1150 and 1151 was the most rapidly phosphorylated and could be recovered as mono-, di- and triphosphorylated peptides cleaved by trypsin at Arg-1143 and either Lys-1153 or Lys-1156. Multiple phosphorylation of this domain appears to partially inhibit the cleavage at Lys-1153 by trypsin. 3. In a second domain (domain 3) containing two phosphorylated tyrosine residues at positions 1316 and 1322 the tyrosines were phosphorylated more slowly than those in domain 2. This domain is close to the C-terminus of the beta-subunit polypeptide chain. 4. At least two further tyrosine residues appeared to be phosphorylated after those in domains 2 and 3. These residues probably residue within a domain lying in close proximity to the inner face of the plasma membrane containing tyrosines 953, 960 and 972, but conclusive evidence is still required. 5. The two-dimensional thin-layer analysis employed in this study to investigate insulin-receptor phosphorylation has several advantages over previous methods based on reverse-phase chromatography. It allows greater resolution of 32P-labelled tryptic peptides and, when coupled to radioautography, is considerably more sensitive. The approach can be readily adapted to study phosphorylation of the insulin receptor within intact cells.

Sign in / Sign up

Export Citation Format

Share Document