Mass spectrometric determination of the amino acid sequence of peptides and proteins

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

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confidence: 99%

Elimination of water from the carboxyl group of GlyGlyH⁺

Balta

Avi̇yente

Lifshitz

2003

“…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.…”

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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...

“…At E Ͼ 95 kcal mol Ϫ1 , the dominant product ion becomes [b 2 Ϫ H] •؉ -2; this dissociation (k B4 ) has a positive activation entropy (⌬S ‡ 298 ) of 2.9 cal mol Ϫ1 K Ϫ1 . By contrast, the similar dissociation from C (k B6 ) has a negative ⌬S ‡ 298 ϭ Ϫ1.7 cal mol Ϫ1 K Ϫ1 , as did tautomerization from B to C (k B9 , ⌬S ‡ 298 ϭ Ϫ0.1 cal mol Ϫ1 K Ϫ1 ) and that from C to B (k B10 , ⌬S ‡ 298 ϭ Ϫ9.5 cal mol Ϫ1 K Ϫ1 ).…”

Section: Direct Hydrogen-atom Migration Between Two ␣-Carbonsmentioning

confidence: 99%

“…C haracterization and identification of a protein by means of mass spectrometry typically rely on fragmentation behaviors of its constituent fragment peptide ions in the gas phase [1][2][3]. Peptides are ionized by protonation via soft-ionization techniques: electrospray ionization [4] and matrix-assisted laser desorption/ionization [5].…”

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confidence: 99%

“…The mechanisms by which protonated peptides dissociate have, therefore, generated much interest [11,12]. Charge-driven amide bond cleavages are induced by the mobile proton, giving b-or y-type ions, which are, respectively, the N-or C-terminal fragments [1,2]. By contrast, open-shell peptide radical cations produced in electron-capture [13][14][15] or electron-transfer dissociation experiments [16] give c-or z-type ions, by cleaving the N-C ␣ bond adjacent to the aminoketyl radical being formed [17][18][19][20].…”

mentioning

confidence: 99%

See 1 more Smart Citation

Kinetics for tautomerizations and dissociations of triglycine radical cations

Siu

Zhao

Laskin

et al. 2009

[7,8], infrared multiphoton dissociation (IRMPD) [9], and blackbody radiation [10]. The mechanisms by which protonated peptides dissociate have, therefore, generated much interest [11,12]. Charge-driven amide bond cleavages are induced by the mobile proton, giving b-or y-type ions, which are, respectively, the N-or C-terminal fragments [1,2]. By contrast, open-shell peptide radical cations produced in electron-capture [13][14][15] or electron-transfer dissociation experiments [16] give c-or z-type ions, by cleaving the N-C ␣ bond adjacent to the aminoketyl radical being formed [17][18][19][20]. The substantial fragmentation differences between protonated and radical cationic peptide provide useful complementary peptide sequencing information [21][22][23]. Ultraviolet (UV) photodissociation has also been employed for peptide fragmentation [24]. UV photodissociation of a peptide/protein chemically modified to incorporate an appropriate chromophore can generate a radical cation that undergoes site-specific, radical-driven dissociations, which are useful in protein identifications [25,26]. Peptide radical cations have also been generated via low-energy CID of a ternary metal complex containing the peptide and auxiliary ligands [27][28][29][30][31][32][33][34][35][36][37]. These methodologies open new avenues whereby the fragmentation chemistries of peptide radical cations can be examined and exploited.Dissociation at an amino-acid side chain is commonplace in the CID of peptide radical cations. These fragmentations are useful in providing differentiating signatures for isobaric residues present [14, 29, 38 -40], e.g., between leucine and isoleucine [14,29], and between aspartic acid and isoaspartic acid [38,39]. For aromatic amino acid residues, cleavage of the C ␣ -C ␤ bond eliminates pquinomethide from tyrosine and 3-methylene indolenine from tryptophan, yielding a glycyl radical with the unpaired electron located on the ␣-carbon [12,27,28,30]. Such ␣-centered radicals have been identified in some anaerobic enzymes [41][42][43] [50]. A radical transfer from the thiyl group in the cysteine residue to a neighboring glycine residue by ␣-hydrogen-atom abstraction generates an ␣-centered radical [51,52], which is stabilized by -electron delocalization between the adjacent electron-withdrawing carbonyl group and the electron-donating amide nitrogen. This stabilization is known as the captodative effect [53]. The intrinsic stability of an amino-acid captodative radical has recently been verified by IRMPD spectroscopy on the histidine radical cation in the Address reprint requests to Professor K