Influence of pigments and protein aging on protein identification in historically representative casein-based paints using enzyme-linked immunosorbent assay.

A systematic study on the influence of pigments and sample aging on casein identification was performed on 30 reconstructed paints. The protein in all the paints was extracted into solution for analysis. The amount of protein that can be retrieved for solution-based analysis in each of the reconstructed paints was studied with a well-developed NanoOrange method before and after artificial aging. The results showed that in the paints with calcium phosphate (in bone black) and copper carbonate, hydroxide, or acetate (in verdigris and azurite), the amount of protein that can be retrieved for liquid-phase analysis is much smaller than the other paints, indicating that the protein degradation was accelerated significantly in those paints. Carbon (in vine black), calcium carbonate (in natural chalk), and calcium sulfate (terra alba gypsum and ground alabaster) did not affect much the amount of protein that can be retrieved in the paints compared to non-pigmented binder, meaning that the protein degradation rate was not affected much by those pigments. Artificial aging was observed to decrease the amount of retrievable protein in all the reconstructed paints that were studied. The enzyme-linked immunosorbent assay (ELISA) method was applied to the 28 reconstructed paints (except two verdigris paints) to assess the protein identification. The ELISA responses from the different paints were compared at fixed protein concentrations. Natural chalk, bone black, raw sienna, stack lead white, and cochineal red-violet lake had the lowest ELISA signal in this study, which indicated that the binding sites (epitopes) on the target protein in these paints are likely to deteriorate more than those in the other paints. Artificial aging did not influence the ELISA response as much as the pigments when the protein concentration was kept the same for the paints that were studied.

Solution dependence of the collisional activation of ubiquitin [M + 7H](7+) ions.

The solution dependence of gas-phase unfolding for ubiquitin [M + 7H](7+) ions has been studied by ion mobility spectrometry-mass spectrometry (IMS-MS). Different acidic water:methanol solutions are used to favor the native (N), more helical (A), or unfolded (U) solution states of ubiquitin. Unfolding of gas-phase ubiquitin ions is achieved by collisional heating and newly formed structures are examined by IMS. With an activation voltage of 100 V, a selected distribution of compact structures unfolds, forming three resolvable elongated states (E1-E3). The relative populations of these elongated structures depend strongly on the solution composition. Activation of compact ions from aqueous solutions known to favor N-state ubiquitin produces mostly the E1 type elongated state, whereas activation of compact ions from methanol containing solutions that populate A-state ubiquitin favors the E3 elongated state. Presumably, this difference arises because of differences in precursor ion structures emerging from solution. Thus, it appears that information about solution populations can be retained after ionization, selection, and activation to produce the elongated states. These data as well as others are discussed.

Oscillations of chiral preference in proline clusters.

Ion mobility/mass spectrometry techniques are used to study the chiral preferences of small proline clusters (containing 2 to 23 proline monomers) produced by electrospray ionization. By varying the composition of the electrospray solution from enantiomerically pure (100% L or 100% D) to racemic (50:50 L:D), it is possible to delineate which cluster sizes prefer homochiral (resolved) or heterochiral (antiresolved) compositions. The results show a remarkable oscillation in chiral preference. Singly protonated clusters, [xPro+H](+) (where x corresponds to the number of prolines), favor homochiral assemblies (for x = 4, 6, 11 and 12); heterochiral structures are preferred (although the preferences are not as strong) for x = 5 and 7. Larger, doubly protonated clusters [xPro+2H](2+) favor homochiral assemblies for x = 18, 19, and 23 and heterochiral structures for x = 14, 16, 17, 20, 21, and 22. Some of the variations that are observed can be rationalized through simple structures that would lead to especially stable geometries. It is suggested that some antiresolved clusters, such as [22Pro+2H](2+), may be comprised of resolved D- and L-proline domains.

Chirality and packing in small proline clusters.

The chiral composition of amino acid clusters may be related to the origin of chirality in biological systems. Here, we use ion mobility/mass spectrometry techniques to investigate the gas phase structures of singly charged proline clusters containing two to six monomers. Using deuterated L-proline (L(D7)) and different electrospray solution compositions varying from enantiopure (50:50 L:L(D7)) to racemic (50:50 L(D7):D), it is possible to study collision cross sections of L-, D-, and mixed xL:xD-proline clusters (where x refers to the number of monomers). These results show that [2Pro+H](+) and [3Pro+H](+) clusters, previously shown (Holliday et al. J. Phys. Chem. A 2012, DOI: 10.1021/jp302677n) to have a very small heterochiral preference, have similar collision cross sections for homochiral and heterochiral proline assemblies. The [4Pro+H](+) and [6Pro+H](+) clusters that exhibit homochiral preference have smaller collision cross sections for homochiral clusters and larger collision cross sections for heterochiral clusters. The [5Pro+H](+) cluster with heterochiral preference has a smaller collision cross section for its heterochiral compositions than for its homochiral compositions. These results suggest that the packing efficiency of subunits within each cluster influences the stability and prevalence of proline multimers as either homochiral or mixed L- and D- clusters.

Collisional activation of [14Pro+2H]2+ clusters: chiral dependence of evaporation and fission processes.

Ion mobility/mass spectrometry techniques are used to investigate the dissociation of the small proline cluster [14Pro+2H](2+) produced by electrospray ionization. While this cluster is known to prefer heterochiral compositions (i.e., mixed L- and D-compositions, J. Phys. Chem. A, submitted for publication), it is possible to produce homochiral forms by electrospraying solutions containing only L- or D-proline. Differences in the measured cross sections for [14Pro+2H](2+) produced from enantiomerically pure (100% l or 100% d) or racemic (50:50 l/d) solutions indicate that homochiral and heterochiral clusters have different structures. Upon low-energy collisional activation, both the heterochiral and homochiral doubly charged structures evaporate neutral proline monomers, resulting in the formation of [xPro+2H](2+) ions (where x = 9-13). At higher activation energies, there is evidence that these smaller clusters (primarily [10Pro+2H](2+)) fission to produce [xPro+H](+) (where x = 1-6). Analysis of product ion intensities reveals a strong chiral preference associated with fissioning. Products of evaporation also show a chiral dependence but to a lesser extent.

Transitions between elongated conformations of ubiquitin [M+11H]11+ enhance hydrogen/deuterium exchange.

Hydrogen/deuterium (H/D) exchange reactions between different elongated conformations of [M + 11H](11+) ions of ubiquitin and D(2)O are studied by a combination of ion mobility spectrometry (IMS) and mass spectrometry techniques. Three conformers (B, C, and D), resolved in the IMS separation, each exchange ∼27 hydrogens upon exposure to 0.06 Torr of D(2)O vapor for ∼35 to 40 ms. However, a region of the IMS spectrum that appears between the C and D states (corresponding to ions that undergo a structural transition during the mobility separation) undergoes substantially more exchanges (∼39 total sites, 44% more than the B, C, and D states). Selection and activation of the individual B, C, and D states reveals that the increased H/D exchange occurs during the transition between structures. Overall, these studies suggest a key process in establishing the maximum exchange levels involves structural transitions, which allow protected sites to be exposed for some fraction of the reaction time. Analysis of changes in exchange levels upon structural transitions can provide insight about common regions of structure that exist in the B, C, and D conformations.