Infrared Spectroscopy of Fragments of Protonated Peptides: Direct Evidence for Macrocyclic Structures of b₅ Ions

Abstract

b ions are of fundamental importance in peptide sequencing using tandem mass spectrometry. These ions have generally been assumed to exist as protonated oxazolone derivatives. Recent work indicates that medium-sized b ions can rearrange by head-to-tail cyclization of the oxazolone structures generating macrocyclic protonated peptides as intermediates. Here, we show using infrared spectroscopy and density functional theory calculations that the b(5) ion of protonated G(5)R exists in the mass spectrometer as an amide oxygen protonated cyclic peptide rather than fleetingly as a transient intermediate. This assignment is supported by our DFT calculations which show this macrocyclic isomer to be energetically preferred over the open oxazolone form despite the entropic constraints the cyclic form introduces.

Towards Understanding the Tandem Mass Spectra of Protonated Oligopeptides. 2: The Proline Effect in Collision-Induced Dissociation of Protonated Ala-Ala-Xxx-Pro-Ala (Xxx = Ala, Ser, Leu, Val, Phe, and Trp)

“…(Only the amide bond introduced by ring formation can be cis or trans). This constrain is not valid any longer for larger b n ions (n≥5) [24][25][26]. However, entropic factors can hinder the corresponding b n -y m _cyclic_peptide pathways while such constraints do not exist for the corresponding 'oxazolone' b n -y m pathways [3,9].…”

“…For these reasons, the b n -y m _cyclic_peptide pathways are usually less active than the corresponding 'oxazolone' b n -y m channels [3,9]. It was, however, recently demonstrated [24][25][26] that the cyclic peptide isomers of the larger b n ions (n≥5) can easily be formed from oxazolone-terminated b n isomers by nucleophilic attack of the N-terminal amine on the protonated oxazolone ring. The third type of rearrangement pathways involves amide bond cleavage initiated by side chain nucleophiles [27][28][29][30][31].…”

Occurrence of C-Terminal Residue Exclusion in Peptide Fragmentation by ESI and MALDI Tandem Mass Spectrometry

“…Such ions formed by rearrangement and multiple bond cleavages disturb the MS/MS spectra and may even lead to incorrect sequence assignment. One illustration of such detrimental behavior is known as peptide scrambling by means of cyclization and reopening of b n ions [12,13]. The extent of such nondirect sequence fragment ion formation and the impact of these sequence permutations on peptide …”

“…However, many MS/MS spectra remains unassigned by sequencing algorithms due to the formation of abundant peaks that do not belong to the direct sequence ion series and related structures (internal and immonium ions, loss of H 2 O, NH 3 ). Many complex rearrangements have been evidenced to explain such nondirect sequence ion production [6][7][8][9][10][11][12], the scrambling effect being recently one of the most studied phenomena [13][14][15][16][17][18][19][20][21][22]. Indeed, these atypical peptide fragmentations deserve great attention in the search for more comprehensive dissociation pathways suitable for efficient automated MS/MS spectra interpretation.…”

“…Despite the great interest raised by the scrambling phenomenon [12][13][14][15][16][17][18][19][20][21][22], less attention has been devoted in the proteomics community to the presence of fragment ions that are not produced by the conventional b n -y m backbone amide pathway but that retain the primary sequence information. As such, one can mention, first, nonspecific fragmentation by the loss of neutral molecules (mainly H 2 O, NH 3 , CO, …) [61,62], and second, the production of internal fragment ions [63,64], especially when residues such as proline, histidine, and arginine were present in the sequence [23,24].…”