Deuterium Labeling of Isoaspartic and Isoglutamic Acids for Mass Spectrometry Analysis

Abstract

Isoaspartic acid (isoAsp) is a common protein modification that spontaneously arises from asparagine or aspartic acid and has been linked to various diseases and health conditions. However, current methods for identifying isoAsp sites in proteins often suffer from ambiguity and have not gained widespread adoption. We developed a novel method that exclusively labels isoAsp with deuterium. This method capitalizes on the unique structural characteristics of isoAsp residues, which possess a free α-carboxyl group and can form an oxazolone ring. Once the oxazolone ring forms, it facilitates racemization at the C^α-position, incorporating a deuteron from a D₂O solvent. The sites of deuterium-incorporated isoAsp in proteins can be unequivocally determined by comparing the precursor and product ion masses of the peptides from proteins reacted in H₂O and D₂O. The effectiveness of this method has been demonstrated through its application to model proteins lysozyme and rituximab. Furthermore, we have confirmed that the isoAsp deuterium-labeling reaction efficiently labels both l- and d-isoAsp without distinction, as well as isoglutamic acid (isoGlu), for which no effective detection methods currently exist.

Scheme 1. Pathways for Spontaneous Deamidation, Isomerization, and Racemization of l-Asn and l-Asp Residues in Proteins

Deamidation of l-Asn begins with the backbone nitrogen of the C-terminal neighboring residue attacking the side-chain carbonyl carbon of Asn, forming succinimide intermediate I. Hydrolysis of intermediate I leads to l-Asp or l-isoAsp. The intermediate I can also undergo racemization via its enol form II, which produces the D configuration of succinimide intermediate III. Hydrolysis of intermediate III yields d-Asp or d-isoAsp. Isomerization and racemization of l-Asp begin similarly with the backbone nitrogen of the C-terminal neighboring residue attacking the side-chain carbonyl carbon of Asp, forming succinimide intermediate I. Hydrolysis of intermediate I leads to either the original l-Asp or l-isoAsp. l-Asp can also be converted to d-isoAsp or d-Asp via intermediates II and III as l-Asn.

Scheme 2. Deuterium Labeling of isoAsp and isoGlu

In the presence of pyridine and acetic anhydride, acetylpyridinium is formed, which then reacts with the α-carboxyl group of isoX (X = Asp when n = 1; X = Glu when n = 2) to form mixed anhydride IV. The α-hydrogen (α-H) of V_I in the keto form is abstracted by pyridine in step a₁ to form an enolate anion represented by two resonance hybrids V_II and V_III. The subsequent reaction of this enolate with deuterium (red) in D₂O in step a₂ gives a racemic oxazolone V_IV incorporating a deuteron at the α-position. The yield of deuterium labeling is significantly enhanced by the repetitive process of generating an oxazolone V_I from IsoX through step b, as long as a large excess of acetic anhydride remains in the reaction mixture. The labile amide, hydroxy, and carboxy protons (blue) being exchanged with deuterons in D₂O are exchanged back to protons in H₂O, but the deuterons incorporated into the α-position of isoX remain unexchanged.

Figure 1

(a) LC–MS analysis of isoDG, DG, and NG subjected to the deuterium-labeling reaction. Each peptide was dissolved in 40 μL of D₂O/pyridine (1:3, v/v) or H₂O/pyridine (1:3, v/v) and reacted with 30 μL of acetic anhydride (Ac₂O) at room temperature for 30 min. After the reaction, the peptide was dried and further incubated in 50 μL of a hydrazine solution (0.35 wt % in H₂O) at room temperature for 30 min. The resulting peptide was then analyzed by LC–MS. The total ion chromatograms (m/z 300–1800) and mass spectra of the three peptides reacted in D₂O (red) or H₂O (blue) are shown. (b) MS/MS spectra of isoDG reacted in D₂O (upper MS/MS spectrum, precursor ions: m/z 663.3, z = 1) or H₂O (lower MS/MS spectrum, precursor ions: m/z 662.3, z = 1). The fragment ions that contain a deuterated isoAsp residue are indicated with an asterisk.

Figure 2

LC–MS/MS analyses of aged and unaged lysozyme subjected to the predigestion isoAsp-labeling reaction. Aged and unaged lysozymes were dissolved in 120 μL of D₂O/pyridine (1:3, v/v) or H₂O/pyridine (1:3, v/v) and reacted with 90 μL of acetic anhydride (Ac₂O) at room temperature for 30 min. After the reaction, the mixture was dried and further incubated in 50 μL of a hydrazine solution (0.35 wt % in H₂O) at room temperature for 30 min. Subsequently, the protein was digested by chymotrypsin and analyzed by LC–MS/MS. (a) Extracted ion chromatograms (m/z 875.89–877.91) encompassing the doubly charged ions of the following three peptides: peak 1: CAKKIVSDGNGMNAW, peak 2: CAKKIVSDGDGMNAW, and peak 3: CAKKIVSDG^isoDGMNAW. (b) Mass spectra of peaks 1–3 from the lysozyme reacted in D₂O or H₂O. (c) MS/MS spectra of peak 3 from the aged lysozyme reacted in D₂O or H₂O. The fragment ions incorporating a deuterium atom are indicated with an asterisk.

Figure 3

Representative LC–MS/MS data for one of the isoAsp sites identified in rituximab. Aged and unaged rituximab were dissolved in 120 μL of D₂O/pyridine (1:3, v/v) or H₂O/pyridine (1:3, v/v) and reacted with 90 μL of acetic anhydride (Ac₂O) at room temperature for 30 min. After the reaction, the mixture was dried and further incubated in 50 μL of a hydrazine solution (0.35 wt % in H₂O) at room temperature for 30 min. Subsequently, the protein was digested by chymotrypsin and analyzed by LC–MS/MS. (a) Extracted ion chromatograms (m/z 664.29–666.31) encompassing the doubly charged ions of the following three peptides containing amino acid residue 55 of the heavy chain of rituximab: peak 1: IGAIYPGNGDTSY, peak 2: IGAIYPGDGDTSY, and peak 3: IGAIYPG^isoDGDTSY. Residue 55 is underlined. (b) Mass spectra of peaks 1–3 from rituximab reacted in D₂O or H₂O. (c) MS/MS spectra of peak 3 from aged rituximab reacted in D₂O or H₂O. The fragment ions incorporating a deuterium atom are indicated with an asterisk.

Figure 4

(a) LC–MS analysis of isoEG, EG, and QG peptides subjected to the deuterium-labeling reaction. Each peptide was dissolved in 40 μL of D₂O/pyridine (1:3, v/v) or H₂O/pyridine (1:3, v/v) and reacted with 30 μL of acetic anhydride (Ac₂O) at room temperature for 30 min. After the reaction, the peptide was dried and further incubated in 50 μL of a hydrazine solution (0.35 wt % in H₂O) at room temperature for 30 min. The resulting peptide was then analyzed by LC–MS. The total ion chromatograms (m/z 300–1800) and mass spectra of the three peptides reacted in D₂O (red) or H₂O (blue) are shown. (b) MS/MS spectra of isoEG reacted in D₂O (upper MS/MS spectrum, precursor ions: m/z 773.3, z = 1) or H₂O (lower MS/MS spectrum, precursor ions: m/z 772.3, z = 1). The m/z values of the fragment ions containing deuterated isoGlu residues in the upper spectrum are indicated with an asterisk.