Electron capture dissociation distinguishes a single D-amino acid in a protein and probes the tertiary structure

Abstract

First results are reported on the application of ECD in analysis of 2ϩ and 3ϩ ions of stereoisomers of Trp-cage (NLYIQWLKDGGPSSGRPPPS), the smallest and fastest-folding protein, which exhibits a tightly folded tertiary structure in solution. The chiral recognition based on the ratios of the abundances of z 18 and z 19 fragments in ECD of 2ϩ ions was excellent even for a single amino acid (Tyr) D-substitution (R chiral ϭ 8.6). The chiral effect decreased with an increase of temperature at the electrospray ion source, as well as at a higher degree of ionization, 3ϩ ions (R chiral ϭ 1.5). A general approach is suggested for charge localization in nϩ ions by analysis of ECD mass spectra of (n ϩ 1)ϩ ions. Application of this approach to 3ϩ Trp-cage ions revealed the protonation probability order in 2ϩ ions: Arg 16 ӷ Gln 5 ϾϷ N-terminus. The ECD results for native form of the 2ϩ ions favor the preservation of the solution-phase tertiary structure, and chiral recognition through the interaction between the charges and the neutral bond network. Conversely, ECD of 3ϩ ions supports the dominance of ionic hydrogen bonding which determines a different gas-phase structure than found in solution. Vibrational activation of 2ϩ ions indicated greater stability of the native form, but the fragmentation patterns did not provide stereoisomer differentiation, thus underlying the special position of ECD among other MS/MS fragmentation techniques. Further ECD studies should yield more structural information as well as quantitative single-amino acid d/l content measurements in proteins. (J Am Soc Mass Spectrom 2004, 15, 1087-1098

Periodic sequence distribution of product ion abundances in electron capture dissociation of amphipathic peptides and proteins

“…ECD FT-ICR MS of six doubly protonated horse myoglobin tryptic fragments ( [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16], [17][18][19][20][21][22][23][24][25][26][27][28][29][30][31], [32][33][34][35][36][37][38][39][40][41][42] Figure S2) and with vibrational activation ( Figure 5) confirm preferential and sometimes periodic product ion formation from peptides with mainly ␣-helical or ␤-turn secondary structure in solution (before protein digestion). Predominantly z-ions are observed because of preferential charge retention at the C-terminal Lys or Arg basic residue upon electron capture, as expected for doubly charged tryptic peptides.…”

“…Supplementary Figure S2 demonstrates preferential cleavage at the NOC ␣ bond number 4 (z nϪ3 ) for fragments with a distinct ␣-helical or turn structure at the N-terminus (fragments [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16], [17][18][19][20][21][22][23][24][25][26][27][28][29][30][31] -118]). The periodicity is more pronounced in AI-ECD data ( Figure 5), demonstrating a period of 3 amino acids for the specified myoglobin fragments.…”

“…Following this approach, Scheme 2 shows preferential ECD product ion formation projected onto the idealized ␣-helical net for myoglobin tryptic fragment [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16].…”

“…The displacement in the central part of the ␣-helix is toward a similarly hydrophilic amino acid. Preferential product ion yield shift toward a less hydrophilic amino acid in the N-terminal part of myoglobin [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16] in AI-ECD is yet to be rationalized. The single amino acid resolution distribution of hydrophobicity along the peptide sequence in amphipathic peptides AA9 and LL9 ( Figure 3) and antimicrobial peptides, piscidins ( Figure 4 Supplementary Figure S1), confirms these observations.…”

“…In ECD/ETD, interaction of a multiply charged peptide or protein ion with an electron/anion results in cleavage of NOC ␣ bonds along the peptide backbone, including chargesite-remote backbone cleavages [2]. ECD is useful for peptide sequence tag generation on a chromatographic timescale for protein identification in bottom-up mass spectrometry [6], de novo peptide sequencing [7], recognition of amino acid chirality [8], location and structural determination of single and multiple posttranslational modifications (PTMs) in peptides [9,10], sequencing and PTM analysis of large proteins (up to 50 -60 kDa) [11], and providing insight into protein secondary and tertiary structures in the gas phase [12,13]. ETD has already demonstrated high potential in several ECD application areas and continues to gain attention in peptide and protein structural analysis, especially in (phospho)proteomics [5,14,15].…”