Naturally Occurring Epsilon Gamma Glutamyl Lysine Isopeptide Crosslinks in Human Neuroblastoma SH-SY5Y Cells

Abstract

Zero-length isopeptide crosslinks between the side chains of glutamine and lysine are the product of transglutaminase activity. It is generally accepted that transglutaminase activity is dormant under physiological conditions because the calcium concentration inside cells is too low to activate transglutaminase to an open conformation with access to the catalytic triad. Traditional assays for transglutaminase activity measure incorporation of biotin pentylamine or of radiolabeled putrescine in the presence of added calcium. In this report, we identified naturally occurring isopeptide crosslinked proteins using the following steps: immunopurification of tryptic peptides by binding to anti-isopeptide antibody 81D1C2, separation of immunopurified peptides by liquid chromatography–tandem mass spectrometry, Protein Prospector database searches of mass spectrometry data for isopeptide crosslinked peptides, and manual evaluation of candidate crosslinked peptide pairs. The most labor intense step was manual evaluation. We developed criteria for accepting and rejecting candidate crosslinked peptides and showed examples of MS/MS spectra that confirm or invalidate a possible crosslink. The SH-SY5Y cells that we examined for crosslinked proteins had not been exposed to calcium and had been lysed in the presence of ethylenediaminetetraacetic acid. This precaution allows us to claim that the crosslinks we found inside the cells occurred naturally under physiological conditions. The quantity of crosslinks was very low, and the crosslinked proteins were mostly low abundance proteins. In conclusion, intracellular transglutaminase crosslinking/transamidase activity is very low but detectable. The low level of intracellular crosslinked proteins is consistent with tight regulation of transglutaminase activity.

Mass Spectrometry of Putrescine, Spermidine, and Spermine Covalently Attached to Francisella tularensis Universal Stress Protein and Bovine Albumin

“…The intervals between the principal, singly charged peaks generally will be equal to the dehydro masses of amino acids, where a dehydro mass is the mass of the intact amino acid minus water. Lists of dehydro amino acid masses can be found in Lockridge and Schopfer 2022 supplement [ 10 ]. Care must be taken in locating the principal peaks because ancillary peaks due to loss of amine, water, and CO will also be present.…”

“…Liquid chromatography tandem mass spectrometry (LC-MS/MS) was performed as previously described [ 10 ]. Briefly, peptides were separated on a Thermo RSLC Ultimate 3000 ultra-high-pressure liquid chromatography system (Thermo Scientific) at 36°C.…”

Chlorpyrifos Oxon Activates Glutamate and Lysine for Protein Cross-linking

“…A majority of them are ruled out by applying our criteria for accepting candidate cross-linked peptide pairs, including manual evaluation of MS/MS spectra. Details of our criteria for accepting candidate cross-linked peptide pairs, with an emphasis on manual evaluation, are described. , Figure is an example of a candidate cross-linked peptide pair that was ruled out as a false positive. A first look at the spectrum suggested a real cross-link because there are six cross-link specific ions: green y8 2+ , green y9 2+ , blue y5 2+ , blue y6 2+ , green y7, and green y8.…”

Polyaminated, acetylated and stop codon readthrough of recombinant Francisella tularensis universal stress protein in Escherichia coli

“…Peptide separation was performed with a Thermo RSLC Ultimate 3000 ultra-high pressure liquid chromatography system (Thermo Scientific) at 36°C, as previously described [ 20 ]. Solvent A was 0.1% formic acid in water, and solvent B was 0.1% formic acid in 80% acetonitrile.…”