The interpretation of collision‐induced dissociation tandem mass spectra of peptides

The oxidation of the sulfhydryl group in cysteine to sulfenic acid, sulfinic acid, and sulfonic acid in proteins is important in a number of enzymatic processes. In this study we examined the fragmentation of four peptides containing cysteine, cysteine sulfinic acid (Cys-SO 2 H), and cysteine sulfonic acid (Cys-SO 3 H) in an ion-trap mass spectrometer. Our results show that the presence of a Cys-SO 2 H in a peptide leads to preferential cleavage of the amide bond at the C-terminal side of the oxidized cysteine residue. The results are important for the determination of the site of the cysteine oxidation and might be useful for the sequencing of cysteine-containing peptides. (J Am Soc Mass Spectrom 2004, 15, 697-702)

mentioning

confidence: 99%

Fragmentation of protonated ions of peptides containing cysteine, cysteine sulfinic acid, and cysteine sulfonic acid

Wang

Shetty

Men

et al. 2004

“…been thought to be acylium ions (Scheme 2) [1,2,4]. Later, it has been shown that the acylium ions are not stable and a N-protonated oxazolone structure (Scheme 2) is proposed for b n ions [13][14][15][16][17][18][19][20].…”

mentioning

confidence: 99%

Elimination of water from the carboxyl group of GlyGlyH⁺

Balta

Avi̇yente

Lifshitz

2003

The elimination of water from the carboxyl group of protonated diglycine has been investigated by density functional theory calculations. The resulting structure is identical to the b 2 ion formed in the mass spectrometric fragmentation of protonated peptides (therefore named "b 2 " in this study). The most stable geometry of the fragment ion ("b 2 ") is an O-protonated diketopiperazine. However, its formation is kinetically disfavored as it requires a free energy of 58.2 kcal/mol. The experimentally observed N-protonated oxazolone is 3.0 kcal/mol less stable. The lowest energy pathway for the formation of the "b 2 " ion requires a free energy of 37.5 kcal/mol and involves the proton transfer from the amide oxygen of protonated diglycine to the hydroxyl oxygen. Fragmentation initiated by proton transfer from the terminal nitrogen has also a comparable free energy of activation (39.4 kcal/mol). Proton transfer initiating the fragmentation, from the highly basic terminal nitrogen or amide oxygen to the less basic hydroxyl oxygen is feasible at energies reached in usual mass spectrometric experiments. Amide N-protonated diglycine structures are precursors of mainly y 1 ions rather than "b 2 " ions. In the lowest energy fragmentation channels, proton transfer to the hydroxylic oxygen, bond breaking and formation of an oxazolone ring occur concertedly but asynchronously. Proton transfer to hydroxyl oxygen and cleavage of the corresponding C™O bond take place at the early stages of the fragmentation step, while ring closure to form an oxazolone geometry occurs at the later stages of the transition. The experimentally observed low kinetic energy release is expected to be due to the existence of a strongly hydrogen bonded protonated oxazolone-water complex in the exit channel. Whereas the threshold energy for "b 2 " ion formation (37.1 kcal/mol) is lower than for the y 1 ion (38.4 kcal/mol), the former requires a tight transition state with an activation entropy, ⌬S ‡ ϭ Ϫ1.2 cal/mol.K and the latter has a loose transition state with ⌬S ‡ ϭ ϩ8.8 cal/mol.K. This leads to y 1 being the major fragment ion over a wide energy range. (J

“…Indeed, mass spectrometric de novo sequencing based on the fragmentation of derivitized peptide ions dates back to the late 1960s and on underivatized peptide ions to the early 1980s (reviewed in [2] and [3]). More recently, MS sequencing has been given considerable impetus by the development of ESI and MALDI, ionization techniques whose efficiencies for producing intact peptide ions are far higher than those that were previously available.…”

mentioning

confidence: 99%

“De novo” peptide sequencing by MALDI-quadrupole-ion trap mass spectrometry: A preliminary study

Zhang

Krutchinsky

Chait

2003

Collision-induced dissociation of singly charged peptide ions produced by resonant excitation in a matrix-assisted laser desorption/ionization (MALDI) ion trap mass spectrometer yields relatively low complexity MS/MS spectra that exhibit highly preferential fragmentation, typically occurring adjacent to aspartyl, glutamyl, and prolyl residues. Although these spectra have proven to be of considerable utility for database-driven protein identification, they have generally been considered to contain insufficient information to be useful for extensive de novo sequencing. Here, we report a procedure for de novo sequencing of peptides that uses MS/MS data generated by an in-house assembled MALDI-quadrupole-ion trap mass spectrometer (Krutchinsky, Kalkum, and Chait Anal. Chem. 2001, 73, 5066 -5077). Peptide sequences of up 14 amino acid residues in length have been deduced from digests of proteins separated by SDS-PAGE. Key to the success of the current procedure is an ability to obtain MS/MS spectra with high signal-to-noise ratios and to efficiently detect relatively low abundance fragment ions that result from the less favorable fragmentation pathways. The high signal-tonoise ratio yields sufficiently accurate mass differences to allow unambiguous amino acid sequence assignments (with a few exceptions), and the efficient detection of low abundance fragment ions allows continuous reads through moderately long stretches of sequence. Finally, we show how the aforementioned preferential cleavage property of singly charged ions can be used to facilitate the de novo sequencing process. T he use of mass spectrometry combined with database searching has gained wide acceptance for rapid, sensitive protein identification [1]. This method requires the availability of extensive protein, cDNA, or genomic sequence from the organism of interest (or at least from a closely related species). If such sequence information is not available, as remains the case for the vast majority of extant organisms, it is desirable to utilize de novo peptide sequencing to identify and obtain information about their protein(s). Although Edman sequencing has for a long time been a method of choice for de novo sequencing of peptides and proteins, mass spectrometry (MS) has also been shown to have considerable utility for this purpose.Indeed, mass spectrometric de novo sequencing based on the fragmentation of derivitized peptide ions dates back to the late 1960s and on underivatized peptide ions to the early 1980s (reviewed in [2] and [3]). More recently, MS sequencing has been given considerable impetus by the development of ESI and MALDI, ionization techniques whose efficiencies for producing intact peptide ions are far higher than those that were previously available. Again, both derivitized and underivatized peptides have been used in this newer work. Thus, chemical derivatization has been applied to simplify/enhance the fragmentation spectra and/or to differentiate N-from C-terminal fragment ions [4 -13]. Notable in this regard is the use of 18 O i...