Fig 1.
A) Isopeptide bond between glutamine and lysine formed by the action of the transglutaminase (TG) enzyme with release of ammonia. B) Isopeptide bond between glutamic acid (or aspartic acid) and lysine formed by exposure to organophosphorus chemicals (OP) with release of water. Figure adapted from [18].
Fig 2.
SDS gel stained with Coomassie blue.
The intense blue band at 50 kDa is tubulin. Tubulin accounts for about 85% of the protein in MAP-rich tubulin Sus scrofa. Bands near the top of the gel include microtubule-associated proteins.
Table 1.
Relationship between search tolerance and number of xlink hits.
Fig 3.
Immunopurified isopeptide from MAP-rich tubulin.
Panel A shows that immunopurification on Dynabeads-Protein G-anti-isopeptide 81D1C2 yielded a single peptide eluting at 20.24 min. Panel B is the MS/MS spectrum of the immunopurified, naturally-occurring isopeptide in MAP-rich tubulin. The crosslink is between K368 of Tau and Q112 of NFH. The quadruply-charged parent ion is at 401.96 m/z.
Fig 4.
MS/MS spectrum of the dansyl-aminohexyl-QQIV adduct at K368 of Tau.
This peptide was found in a gel slice from MAP-rich tubulin Sus scrofa that had been treated with dansyl-aminohexyl-QQIV and human transglutaminase. A complete y-ion sequence was obtained from the triply-charged parent ion, at 524.28 m/z. Characteristic ions from the fragmentation of dansyl-aminohexyl QQIV were observed at 170.10, 234.06, 347.14, 364.17, and 475.20 (shown in red) [35]. Linkage between the peptide lysine and the adduct glutamine occurs at both glutamines in Q1Q2IV. The ion at 475.20 is evidence for linkage to Q2. The ion at 1212.60 is evidence for linkage to Q1.
Fig 5.
Chemically synthesized peptides crosslinked by bacterial transglutaminase.
Panel A is an Orbitrap spectrum of the crosslinked product after tryptic digestion. The triply-charged parent ion has a mass of 535.62 m/z. Panel B is a MALDI-TOF/TOF spectrum of the same peptide. The singly-charged parent ion has a mass of 1604.86 m/z.
Fig 6.
MS/MS spectrum of an isopeptide crosslink between K163 of tubulin alpha-1A and E158 of tubulin beta-4B induced by treatment of MAP-rich tubulin Sus scrofa with 100 μM chlorpyrifos oxon.
The parent ion at 566.79 is in charge state +3. Arrows point to 14 crosslink-specific fragment ions. Structures are shown for 8 crosslink-specific fragment ions. Fragment ions from both peptides are present. The Protein Prospector score is 29.8 and the score difference is 6.2. The matched intensity is 47.1%. Unassigned, black peaks could not be fit to any sequence.
Fig 7.
MS/MS spectrum of a diethoxyphospho-adduct on lysine 163 from tubulin alpha-1A.
A). Blue masses define a y-ion sequence. Red masses define a b-ion sequence. Unlabeled blue and red lines represent fragments minus NH3, H2O or CO. The structures at 220.1 and 237.1 m/z represent characteristic ions for diethoxyphospho-lysine. B) Green masses define the b-ion sequence for K(dep)LSVDYGK, the rearranged form of the original peptide.
Fig 8.
Chemically induced protein crosslinking is initiated when chlorpyrifos oxon makes a covalent bond with lysine to make diethoxyphospho-lysine.
The activated diethoxyphospho-lysine reacts with a nearby glutamic acid or aspartic acid to make an isopeptide bond with release of diethoxyphosphate. The KE and KD crosslinks are resistant to trypsin.
Fig 9.
A scheme for possible structures that could be associated with an isopeptide crosslink in a linear peptide sequence.
Fig 10.
A proposed mechanism for cleavage of the isopeptide bond in the mass spectrometer.