Use of 18O labels to monitor deamidation during protein and peptide sample processing

Abstract

Nonenzymatic deamidation of asparagine residues in proteins generates aspartyl (Asp) and isoaspartyl (isoAsp) residues via a succinimide intermediate in a neutral or basic environment. Electron capture dissociation (ECD) can differentiate and quantify the relative abundance of these isomeric products in the deamidated proteins. This method requires the proteins to be digested, usually by trypsin, into peptides that are amenable to ECD. ECD of these peptides can produce diagnostic ions for each isomer; the c. + 58 and z - 57 fragment ions for the isoAsp residue and the fragment ion ((M + nH)((n-1)+.) - 60) corresponding to the side-chain loss from the Asp residue. However, deamidation can also occur as an artifact during sample preparation, particularly when using typical tryptic digestion protocols. With 18O labeling, it is possible to differentiate deamidation occurring during trypsin digestion which causes a +3 Da (18O1 + 1D) mass shift from the pre-existing deamidation, which leads to a +1-Da mass shift. This paper demonstrates the use of (18)O labeling to monitor three rapidly deamidating peptides released from proteins (calmodulin, ribonuclease A, and lysozyme) during the time course of trypsin digestion processes, and shows that the fast (approximately 4 h) trypsin digestion process generates no additional detectable peptide deamidations.

Figure 1

Mass spectra of the triply charged calmodulin peptide (⁹¹VFDKDGNGYISAAELR₁₀₆) extracted at different time from the tryptic digestion solution in H₂¹⁶O (left column) and in H₂¹⁸O (right column). The inset shows the theoretical isotopic distribution of this peptide. The isotopic clusters were assigned in the ¹⁸O spectra as ¹⁸O_m + nD, where m and n indicate the number of ¹⁸O incorporations and deamidations, respectively.

Figure 2

Mass spectra of the doubly charged RNase A peptide (⁶⁷NGQTNCYQSYSTMSITDCR₈₅) extracted at different time from the tryptic digestion solution in H₂¹⁶O (left column) and in H₂¹⁸O (right column). The inset shows the theoretical isotopic distribution of this peptide. The isotopic clusters were assigned in the ¹⁸O spectra in the same way as in figure 1.

Figure 3

Mass spectra of the doubly charged RNase A peptide (⁴⁶NTDGSTDYGILQINSR₆₁) extracted at different time from the tryptic digestion solution in H₂¹⁶O (left column) and in H₂¹⁸O (right column). The inset shows the theoretical isotopic distribution of this peptide. The isotopic clusters were assigned in the ¹⁸O spectra in the same way as in figure 1.

Figure 4

The percentage of each isotopic cluster at different digestion time as calculated using the least squares method for the ¹⁸O labeled: (a) calmodulin tryptic peptide (91-106), (b) RNase A tryptic peptide (67-85), (c) lysozyme tryptic peptide (46-61) and (d) calmodulin tryptic peptide (91-106) triplicate experiments. (a-d) -◆-: ¹⁸O₁, -□-: ¹⁸O₂, -▲-: ¹⁸O₁ + 1D, -○-: ¹⁸O₂ + 1D, except in (b) ¹⁸O₁ + 2D.

Figure 5

ECD spectra of the triply charged calmodulin tryptic peptide (91-106) labeled in H₂¹⁸O at different time points: (a) 2 hr, (b) 24 hr. The insets of (a) show the [M − 60] ion (left) and the cleavage pattern (right). The insets of (b) show the isotopic distributions of the c₆· + 58 (left), [M − 60] (middle), and z₁₀ − 57 (right) ions. *: electronic noise, ω2: harmonics.

Scheme 1

The mechanism of the asparagine residue deamidation in H₂¹⁸O.