Our goal was to synthesize a model crosslinked dipeptide composed of KIETHK linked to QLEAHNR through a KQ isopeptide bond. The model dipeptide could be used to validate the MS/MS spectrum of the dipeptide from MAP-rich tubulin. The plan was to link 2 precursor peptides through the crosslinking action of bacterial transglutaminase, followed by digestion with trypsin to yield the desired KQ dipeptide. Genscript Inc. synthesized precursor peptides RFAGYIDKVRQLEAHNR sample name NFH and ITHVPGGGNRKIETHK sample name Tau. The chemically synthesized peptides, 2 mg each in 200 μL of 20 mM imidazole pH 7.5, were combined into one vial. The pH of the 400 μL peptide solution was raised from 6 to 7 by addition of 4 μL of 1 M sodium hydroxide. The crosslinking reaction was catalyzed by 6 μL of bacterial transglutaminase (27 μg) for 16 h at 37˚C. MALDI-TOF mass spectrometry showed a single peak at the predicted mass of the crosslinked construct, 3800 Da, suggesting that 100% of the peptides had been crosslinked. A 10 μL aliquot containing 50 μg of 3800 Da crosslinked peptide was diluted to 50 μL with water and digested with 2 μg trypsin. The expected peak at 1605 Da, for the KIETHK_QLEAHNR crosslinked product was present at low abundance. The digest was enriched for the 1605 Da peptide by high performance liquid chromatography on a C18 Phenomenex column Prodigy 5 μ ODS(2), 11x 4.60 mm, part no.00D-3300-EO where the 1605 Da peptide eluted in 16% acetonitrile, 0.1% trifluoroacetic acid. The fraction containing the 1605 Da peptide was subjected to LC-MS/MS on the Orbitrap mass spectrometer.

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