High-order structure and dissociation of gaseous peptide aggregates that are hidden in mass spectra

Abstract

Injected-ion mobility and high-pressure ion mobility techniques have been used to examine the conformations of bradykinin, insulin chain A, and several other peptide ions in the gas phase. Under the experimental conditions employed, evidence for multimer formation in the mass spectra of peptides is minimal or absent altogether. However, ion mobility distributions show that aggregates of peptides (containing a single charge per monomer unit) are observed at the same mass-to-charge ratios as the singly charged parent ions. Collision cross sections for these clusters show that they have tightly packed roughly spherical conformations. We have bracketed the average density as 0.87 < p < 1.00 g cm-3. In some cases, specific stable aggregate forms within a cluster size can be distinguished indicating that some high order structures are favored in the gas phase. Multimer formation between different sizes of polyalanine peptides shows no evidence for size specificity in aggregate formation. Collisional and thermal excitation studies have been used to examine structural transitions and dissociation of the multimers. Aggregates appear to dissociate via loss of singly charged monomers. The observation that peptide multimers can be concealed in mass spectral data requires that fragmentation patterns and reactivity studies of singly charged monomers be undertaken with care.

Fragmentation of doubly-protonated peptide ion populations labeled by H/D exchange with CD3OD

“…The ion mobility and H/D exchange of the nonapeptide bradykinin and related analogues have been extensively studied [10,12,[15][16][17][18][19][20][21][22][23][24][25][26][27][28]. Ion mobility measurements have shown that the singly-and doubly-protonated ions of bradykinin have a similar cross-section, while the triplyprotonated ion has a cross-section that is approximately 15% larger, providing evidence for a much less compact structure [15][16][17].…”

“…Ion mobility measurements have shown that the singly-and doubly-protonated ions of bradykinin have a similar cross-section, while the triplyprotonated ion has a cross-section that is approximately 15% larger, providing evidence for a much less compact structure [15][16][17]. H/D exchange studies with D 2 O, CD 3 OD, ND 3 , and DI have detected two non-interconverting ion populations for doubly-and triply-protonated bradykinin [20,22,[24][25][26][27].…”

“…H/D exchange studies with D 2 O, CD 3 OD, ND 3 , and DI have detected two non-interconverting ion populations for doubly-and triply-protonated bradykinin [20,22,[24][25][26][27]. These ion populations were not detected by traditional ion mobility experiments [15][16][17], suggesting the conformers have a similar collisional cross-section. Another possibility is that the conformations are different in the two instruments because of different timescales (milliseconds in ion mobility and minutes in FT-ICR).…”

Further studies on the origins of asymmetric charge partitioning in protein homodimers

“…In contrast, mass spectrometry requires little sample, and with electrospray ionization (ESI) or soft laser desorption/ionization (John Fenn and Kiochi Tanaka 2002 in chemistry), a wide variety of biomolecules and specific complexes can be introduced into a mass spectrometer. A number of methods have been developed to probe the general three-dimensional shapes of biomolecule ions and noncovalent complexes using the techniques of ion mobility [1][2][3][4][5][6][7][8], H/D exchange both in solution [9,10] and the gas phase [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28], and proton-transfer reactivity [29,30].…”

Ion Mobility-Mass Spectrometry with a Radial Opposed Migration Ion and Aerosol Classifier (ROMIAC)

“…The ROMIAC also demonstrates high resolution with the TAAX standards (close to R nd ; Table S4), though resolution of the calibrant peptides and proteins were generally not as high as those of the TAAX standards (Table S6). This reduced resolution for peptides is fairly typical and is often assigned to the existence of multiple conformers within the mobility envelope [12,13,14,15,16,17] Values from [6]. Mobility values are in units of [cm…”