We initiated a study using mass spectrometry to determine if AA sequence differences exist between Ptx and Ptxd when examined from different sources. Using proteins (i.e., Ptx) from strains that may not share the exact same AA sequence or that have undergone different inactivation treatments could result in differences in immune effectiveness. Protein sequence variation between strains may be small (i.e., single AA addition, deletion, or substitution) or potentially more extensive with the addition or deletion of immunogenically relevant motifs or domain structures. As a result, protein sequence variance could lead to subtle structural, conformational, and/or functional differences that can be detected only through the use of sensitive technologies.
Previous MS investigations have been used for identification, differentiation, and characterization of protein complexes. For example, Nandakumar et al. [22
] utilized MS techniques for identification of bacterial proteins. Protein mixtures were subject to tryptic in-solution digestion and analyzed by reverse phase nano LC-MS/MS followed by database analysis. Other investigators have used MALDI-TOF MS/MS for the analysis of peptides generated from the enzymatic cleavage of Clostridium botulinum
C3 coenzyme [23
]. Also, Yoder and colleagues analyzed tryptic digests of Vaccinia virus using MALDI-TOF, as well as a quadrupole ion trap mass spectrometer and a quadrupole TOF to identify proteins associated with infection [24
]. Additionally, nano-LC-MS/MS was used to identify AA variability between two-bovine surfactant proteins [25
]. In our study, an analytical and proteomic evaluation was conducted to assess any composition (i.e., AA sequence of the protein components) and formulation (i.e., how much of a specific component) disparities among Ptx and Ptxd.
Based on AA coverages and peptide detection alone, the two Ptxs and two Ptxds appear to have similar sequences, an indication that they were derived from the same origin, although different purification schemes and inactivation methods most likely were employed. The fact that the Ptx and Ptxd sources shared similar tryptic digest patterns, in terms of the peptides being generated and detected as well as spectrum profiles, suggests homogeneity. The presence of RG improved protein digestion resulting in higher-percent coverages generally across source as well as Ptx subunits. In addition, RG treatment appeared to have exposed the smallest-sized S5, resulting in an increase in coverage for this subunit. In doing so, additional peptides were detected that once again were shown to be similar among Ptx and Ptxd sources based on y- and b-ion distribution and relative intensities of the fragmented peptides. Moreover, studies by Sekura et al. [26
] revealed, by amino acid analysis, that Ptx purified from Bp strain 165 was indistinguishable from Ptx prepared from the well-characterized Bp Tahoma strain. In addition, Tummala et al. confirmed Ptx sequence homogeneity among their Bp culture supernatant purified Ptx as compared with previously reported sequence information [19
]. These data are confirmatory and strengthen the MS results in this study for Ptx sequence homogeneity. However, although the data suggest protein sequence homogeneity, if the sequence is indeed different but not present in the database then, upon searching, the generated peptides would not be detected resulting in a lower peptide map coverage.
Ptx is a major virulence factor and in its natural state has physiological inhibitory effects [11
]. The subunits are highly immunoreactive, including the dominant S1, making it an ideal component for vaccines. To reduce the cellular lethality induced by Ptx, the Ptx is inactivated or detoxified in its final vaccine form. Inactivation occurs by chemical modification (i.e., formaldehyde or glutaraldehyde) via the formation of covalent crosslinkings on targeted AA (usually lysines). Interestingly in Ptxd samples, the oligomeric containing subunits percent AA coverage was lower. The S2–S5 subunits, rich in lysine are the subunits that are known to be heavily modified due to the chemical action exhibited by formaldehyde and/or glutaraldehyde [13
]. As a result of modification, these peptides and subsequent MS-MS profiles would thus not be detected using traditional selected database searching tools resulting in lower AA coverages.
Although formaldehyde, for instance, is an ideal reagent for chemical crosslinking, it also immobilizes the antigen (i.e., Ptx) and alters its conformation preventing a natural interaction between the antibody and antigen. In other words, formaldehyde reduces the accessibility of antibodies to their epitopes by creating steric hindrance or by altering the epitope site itself. In Ptxd samples, inactivation by formaldehyde appears to have a severe impact on peptide detection, due to crosslinking of lysine on the Ptx subunits [13
]. These subunits are most likely masked preventing trypsin, whose natural recognition sites are lysine and arginine, to effectively cleave the protein resulting in a lower AA coverage. On a cellular level, it is sensible for these subunits to be inactivated heavily, since in vivo, they make up the oligomeric complex that adheres to the human host cells which upon binding to the appropriate receptor initiates Ptx function. Blocking these cellular activities, in essence, inhibits or diminishes further downstream enzymatic activity by S1, which would allow for progression of normal immune responses by the host towards Bp. Besides, S1 has consistently had higher AA coverage reporting among all sources, which could be explained by the fact that S1 lacks lysines. Although S1 is deficient in lysine, free cellular lysine in the presence of formaldehyde would still create crosslinking bridges that lead to the detoxification [28
] but to a lesser extent compared to S2–S5. As a result of minimal chemical bombardment and thus potentially incomplete inactivation by formaldehyde on the toxic S1 component, residual toxicity may remain, with varying levels among Ptxd sources, could lead and be an explanative factor for differential immunity, albeit speculative.