Abstract
Bacterial transglutaminase was used to label human plasma proteins with fluorescent tags. Protein lysines were modified with dansyl-epsilon-aminohexyl-Gln-Gln-Ile-Val-OH (dansylQQIV), while protein glutamines were modified with dansyl cadaverine. Labeled proteins included human butyrylcholinesterase, apolipoprotein A-1, haptoglobin, haptoglobin-related protein, immunoglobulin heavy chain, and hemopexin. Tryptic peptides were analyzed by LC-MS/MS on an Orbitrap Fusion Lumos mass spectrometer. Modified residues were identified in Protein Prospector and Proteome Discoverer searches of mass spectrometry data. The MS/MS fragmentation spectra from dansylQQIV-modified peptides gave intense peaks at 475.2015, 364.1691, 347.1426, 234.0585, and 170.0965 m/z. These signature ions are useful markers for identifying modified peptides. Human butyrylcholinesterase retained full activity following modification by dansylQQIV or dansyl cadaverine.
Highlights
Protein-to-protein crosslinking reactions can be catalyzed by transglutaminase [1]
Transglutaminase catalyzes the formation of an isopeptide bond between the epsilon amino group of lysine and the gamma glutamyl group
These are consistent with fragments from the dansylQQIV moiety
Summary
Protein-to-protein crosslinking reactions can be catalyzed by transglutaminase [1]. The side chain of glutamine makes a transient bond with the active site cysteine of transglutaminase accompanied by the loss of a molecule of ammonia [2], followed by reaction with the side chain of lysine to produce the epsilon-gamma-glutamyl lysine isopeptide bond. These fluorescent tags were used to identify eight glutamines and 10 lysines in human Tau protein [5] susceptible to the crosslinking action of transglutaminase. The commercially available bacterial transglutaminase makes gamma-glutamyl-epsilon-lysine bonds [10,11,12], with the advantage that bacterial transglutaminase requires no calcium, no reducing agent, and is less costly
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