Fragmentation behavior of Amadori-peptides obtained by non-enzymatic glycosylation of lysine residues with ADP-ribose in tandem mass spectrometry

2010 ◽  
pp. n/a-n/a ◽  
Author(s):  
Maria Fedorova ◽  
Andrej Frolov ◽  
Ralf Hoffmann
Keyword(s):  
Mass Spectrometry ◽  
Tandem Mass Spectrometry ◽  
Tandem Mass ◽  
Fragmentation Behavior ◽  
Lysine Residues ◽  
Enzymatic Glycosylation

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.

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