Practical Protein Chemistry - A. Darbre 1989

Application of electron impact mass spectrometry for determining the amino acid sequence of peptides and proteins
Interpretation of mass spectra
Peptide fragmentation pathways

1. The electron-impact fragmentation of permethylated acetyl Peptides is primarily driven by the Cleavage of amide bonds, specifically the C—N bonds (Fig. 19.3). The resulting ions generated by this cleavage directly reflect the aminoacyl sequence of the peptide. The mass differences between such ions correspond to the characteristic masses of individual amino acid residues. First, the mass spectrum is examined to identify the ion corresponding to the N-terminal amino acid residue, whose m/z mass-to-charge ratio typically falls within the range of 114–257. By successively adding the masses of amino acid residues that may comprise the peptide to this base value, one can locate ions with higher mass-to-charge ratios in the spectrum, thereby establishing the Amino Acid Sequence of the test sample. For instance, consider a peptide A-B-C-D, where A, B, C, and D denote modified amino acid residues. Upon electron impact, this peptide yields the ions A⁺, AB⁺, ABC⁺, and ABCD⁺. The potential mass numbers of N-terminal ions typically formed during peptide fragmentation are listed in Table 19.1. It should be noted that mass-spectrometric Determination of the Introduction/19.html">Primary Structure of peptides does not allow for distinguishing between leucine and isoleucine residues.

Class="center">Table 19.1. Masses of N-terminal ions and their corresponding amino acid residues in the peptide chain in electron-impact mass spectra of acetylated permethylated peptides

2. Amino acid residues whose side chains can generally be represented as CH₂X (where the substituent X may contain conjugated bonds) are prone to C—N bond cleavage accompanied by hydrogen atom migration (Fig. 19.4). This pathway is characteristic of peptides containing aspartic acid, asparagine, phenylalanine, Histidine, Tyrosine, and Tryptophan residues. It is worth noting that such fragmentation generates novel peptides whose amino acid sequence begins with the residue that underwent C—N bond cleavage, and the mass spectrum will exhibit peaks corresponding to the ions defining their sequence. The masses of N-terminal peptide ions formed via C—N bond cleavage are summarized in Table 19.2.

FIG. 19.4. N—C cleavage of peptide bonds in permethylated peptides accompanied by hydrogen atom rearrangement.

Table 19.2. Masses of N-terminal ions formed As a result of C—N bond cleavage

3. Glutamic Acid and Glutamine residues, regardless of their position within the chain, typically undergo partial cyclization to yield N-terminal pyrrolidonecarboxylate ions. This cyclization results in the appearance of ion 1 at m/z 98 in the spectrum, whereas the corresponding N-terminal ion 2 at m/z 126 appears as a weak peak or is entirely absent.

4. Side-chain fragmentation of Certain amino acid residues often facilitates the interpretation of peptide mass spectra. Examples include Serine and Threonine residues, whose side chains readily undergo methanol elimination. Additionally, threonine residues are characterized by the loss of the entire side chain via C—C bond cleavage, with or without hydrogen migration. Methionine behaves similarly, with its residue losing a CH₃SH molecule (M 48) and/or the entire side chain.