Surface-induced dissociation shows potential to be more informative than collision-induced dissociation for structural studies of large systems

Abstract

The ability to preserve noncovalent, macromolecular assemblies intact in the gas phase has paved the way for mass spectrometry to characterize ions of increasing size and become a powerful tool in the field of structural biology. Tandem mass spectrometry experiments have the potential to expand the capabilities of this technique through the gas-phase dissociation of macromolecular complexes, but collisions with small gas atoms currently provide very limited fragmentation. One alternative for dissociating large ions is to collide them into a surface, a more massive target. Here, we demonstrate the ability and benefit of fragmenting large protein complexes and inorganic salt clusters by surface-induced dissociation (SID), which provides more extensive fragmentation of these systems and shows promise as an activation method for ions of increasing size. ver the past two to three decades, mass spectrometry (MS) has expanded significantly, from its early use as a technique for measuring the isotopes of elements and analyzing volatile compounds, to a technique that is now routinely used to study nonvolatile molecules and large macromolecular complexes. Increasingly, mass spectrometry and ion mobility/mass spectrometry are described as structural biology tools. Mass spectrometry has recently provided insights on posttranslational modifications [1], mono-and polydisperse subunit stoichiometry [2], subunit organization [3,4], and noncovalent protein-ligand binding sites [5]. In addition to revealing structural information, mass spectrometry can be used to monitor dynamic processes, such as protein complex assembly [6], protein-substrate and protein-protein interactions [7,8], and substratespecific conformational changes [9].One of the limitations of current technology, however, is the fact that commercial instrumentation is still hampered by the amount of dissociation that can be induced from large biomolecular complexes. Often, MS has to be combined with many solution-based experiments (H/D exchange plus digestion, chemical crosslinking plus digestion, limited proteolysis, solution disruption by changes in ionic strength) because the MS instruments commercially available do not provide extensive dissociation of these massive complexes. A typical dissociation result that is achieved is ejection of a monomer subunit as illustrated here (Figure 1) for a small heat shock protein (sHSP) dodecamer, consisting of 12 subunits each weighing 16.9 kDa [8,10,11]. The 16.9 kDa monomer typically carries away a large percentage of the charge of the original complex with the remainder of the charge on the 11-mer that weighs 186 kDa. Investigators have concluded that this behavior, which has been observed by several research groups for many different noncovalent protein complexes, happens because the low-energy collisions with a gas initiate unfolding of one of the protein subunits [12][13][14]. As this subunit unfolds, it gains surface area, thus allowing it to accept more charge. Eventually, when the unfolding protein and the remainde...

Collisional activation of protein complexes: Picking up the pieces

“…Black-body infrared radiative dissociation [15], electron-capture dissociation [17], infrared multiphoton dissociation [14,16], and surface-induced dissociation [18,64] have all been applied to protein assemblies. Of these, the latter is notable for the large amount of energy deposited into the protein complexes in a very short time, resulting in more extensive dissociation than that in CID [65]. Furthermore, small multimeric complexes were shown to dissociate into equally charged monomeric parts, rather than monomers and stripped oligomers [64,65].…”

“…Of these, the latter is notable for the large amount of energy deposited into the protein complexes in a very short time, resulting in more extensive dissociation than that in CID [65]. Furthermore, small multimeric complexes were shown to dissociate into equally charged monomeric parts, rather than monomers and stripped oligomers [64,65]. These differences are rationalized in terms of the collisions being more energetic in the center-of-mass frame and the speed of energy deposition being far greater [65].…”

“…Furthermore, small multimeric complexes were shown to dissociate into equally charged monomeric parts, rather than monomers and stripped oligomers [64,65]. These differences are rationalized in terms of the collisions being more energetic in the center-of-mass frame and the speed of energy deposition being far greater [65]. Efforts have been made to bring these differences to CID, both by using heavier target gases [66] and by increasing the range of acceleration voltages in the collision cell [49,67].…”

Chaperonin Cofactors, Cpn10 and Cpn20, of Green Algae and Plants Function as Hetero-oligomeric Ring Complexes

“…8B, bottom panel), corresponding to the removal of close to the complete C-terminal ϳ10-kDa domain. Such fragmentation of the polypeptide backbone during collisional activation of proteins in the gas phase is unusual, and it is indicative of a particularly labile covalent bond (55). Formation of AtCpn20 mut ⌬C was not observed with free AtCpn20 mut (data not shown) but only in the presence of the chaperonin, suggesting that one domain of an AtCpn20 mut subunit is excluded from interacting with AtCpn60␣ 7 ␤ 7 and is forced to adopt an alternative conformation, thereby rendering it susceptible to fragmentation in the MS measurement.…”

Top-down proteomic analysis of protein pharmaceuticals, mixtures of protein complexes, and ribosomal protein extracts by capillary zone electrophoresis-mass spectrometry

“…With surfaces, the collisional cross-section is significantly greater than for comparatively smaller gas ions, thereby allowing for superior energy transfer [157]. In the context of intact protein analyses, SID has been demonstrated to allow for superior fragmentation of larger species than ECD/ETD [161]. Especially in the context of native protein analysis, applications with SID have demonstrated that individual subunits of a multiprotein complex can be dislodged without subsequent unfolding of the dissociated monomer, as seen with CID/HCD [161].…”