The Negative Ion Mass Spectra of [M-H]− Ions Derived From Caeridin and Dynastin Peptides. Internal Backbone Cleavages Directed Through Asp and Asn Residues

We have previously shown that peptide amide hydrogens undergo extensive intramolecular migration (i.e., complete hydrogen scrambling) upon collisional activation of protonated peptides (Jørgensen et al. J. Am. Chem. Soc. 2005, 127, 2785-2793. The occurrence of hydrogen scrambling enforces severe limitations on the application of gas-phase fragmentation as a convenient method to obtain information about the site-specific deuterium uptake for proteins and peptides in solution. To investigate whether deprotonated peptides exhibit a lower level of scrambling relative to their protonated counterparts, we have now measured the level of hydrogen scrambling in a deprotonated, selectively labeled peptide using MALDI tandem time-of-flight mass spectrometry. Our results conclusively show that hydrogen scrambling is prevalent in the deprotonated peptide upon collisional activation. The amide hydrogens . In comparison, deprotonated peptides have received much less attention, and the mechanistic details of their fragmentation behavior are less well understood. We and others have previously investigated the proton mobility in protonated peptides and proteins upon collision-induced dissociation (CID) [2][3][4][5][6][7][8][9][10][11][12]. Our studies were motivated by earlier reports claiming that CID could be used to determine site-specific incorporation of deuterium in the backbone amides of peptides that were deuterated in solution [13][14][15][16]. In these reports, it was suggested that the level of intramolecular migration of amide hydrogens ( 1 H/ 2 H) in the protonated, collisionally activated peptide was negligible. It is important to emphasize that negligible levels of hydrogen migration would dramatically improve the resolution of localizing deuterated sites by mass spectrometric analysis of peptides and proteins labeled in solution ( 1 H/ 2 H) exchange experiments. But recent results have demonstrated unequivocally that all backbone amide hydrogens are positionally randomized upon collisional activation in the gaseous protonated peptide (i.e., causing 100% hydrogen scrambling) [3,6,7,10]. This implies that protonation of amide nitrogens is a reversible process in the gaseous activated peptide, and that the mobile proton (or deuteron) samples all exchangeable sites before peptide bond cleavage. Interestingly, this also occurs in singly protonated peptides exhibiting a preferred site-selective cleavages at Asp residues, which are thought to be induced by a reduced proton mobility [6]. Deprotonated peptides are deficient in protons and it is thus reasonable to assume that such ions might exhibit a lower degree of hydrogen scrambling than their protonated counterparts. Furthermore, low-energy CID of deprotonated peptides often yield a fragment ion type (c-ion) that is observed only rarely in protonated peptides [17]. The c-ion is the N-terminal fragment ion that results from backbone N-C ␣ bond cleavage. As anionic peptides thus exhibit different fragmentation pathways (relative to their protonated counterparts) and they...

Section: Resultssupporting

confidence: 92%

Hydrogen atom scrambling in selectively labeled anionic peptides upon collisional activation by MALDI tandem time-of-flight mass spectrometry

Bache

Rand

Roepstorff

et al. 2008

“…Penta-alanine also produces a n − and ″b n − , which is typical of oligoalanine peptides in the negative ion mode [41]. Neutral loss peaks common to specific amino acid residues are abundant in the spectra; for example, elimination of H 2 O and CH 2 O from serine [42][43][44][45][46][47][48][49], the guanidino group (HN=C=NH) from arginine [40,49,50], H 2 O from aspartic acid [51][52][53][54], O=C 6 H 4 =CH 2 from tyrosine [54], and CH 3 SH, CH 3 SCH 3 , and CH 2 CH 2 SCH 3 from methionine [55].…”

Section: Resultsmentioning

confidence: 99%

A Comparison of the Effects of Amide and Acid Groups at the C-Terminus on the Collision-Induced Dissociation of Deprotonated Peptides

Bokatzian-Johnson

Stover

Dixon

et al. 2012

The dissociative behavior of peptide amides and free acids was explored using low-energy collision-induced dissociation and high level computational theory. Both positive and negative ion modes were utilized, but the most profound differences were observed for the deprotonated species. Deprotonated peptide amides produce a characteristic c m-2 -product ion (where m is the number of residues in the peptide) that is either absent or in low abundance in the analogous peptide acid spectrum. Peptide acids show an enhanced formation of c m-3 -; however, this is not generally as pronounced as c m-2 -production from amides. The most notable occurrence of an amide-specific product ion is for laminin amide (YIGSR-NH 2 ) and this case was investigated using several modified peptides. Mechanisms involving 6-and 9-membered ring formation were proposed, and their energetic properties were investigated using G3(MP2) molecular orbital theory calculations. For example, with C-terminal deprotonation of pentaglycine amide, formation of c m-2 -and a 6-membered ring diketopiperazine neutral requires 931.6 kcal/mol, which is 26.1 kcal/mol less than the analogous process involving the peptide acid. The end group specific fragmentation of peptide amides in the negative ion mode may be useful for identifying such groups in proteomic applications.

“…Such a result, discussed below in the CID data section, was previously reported by Steinborner and Bowie. [33]. Note that this neutral loss takes place exclusively from the doubly deproto- Ϫ product ions.…”

Section: Negative Ions Generated In the Esi Source And Under Low Collmentioning

confidence: 98%

“…Interestingly, an unexpected base peak was detected at m/z 372, which corresponded to a loss of 44 u from the [Mp Ϫ 2H] 2Ϫ species. Such a 44-u neutral loss is generally attributed to a CO 2 release [33]. However, high resolution analysis of the m/z 372 ion, using the LTQ Orbitrap instrument, permitted to assign unambiguously this neutral loss to an elemental composition of C 2 H 4 O, corresponding to the release of CH 3 CHO from the threonine side chain.…”

Section: Negative Ions Generated In the Esi Source And Under Low Collmentioning

confidence: 99%

“…Negative-ion fragmentations of peptides are often as informative as those generated in positive-ion mode [33]. Here, negative-ion mode fragmentations revealed information complementary to those arising from the positive-ion mode, which allowed mainly the characterization of the peptide moiety.…”

Section: Negative Ions Generated In the Esi Source And Under Low Collmentioning

confidence: 99%

See 1 more Smart Citation

Collision induced dissociation-based characterization of nucleotide peptides: Fragmentation patterns of microcin C7–C51, an antimicrobial peptide produced by Escherichia coli

Petit

Zirah

Rebuffat

et al. 2008

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