5.4.1 Gas Phase OH-Initiated Footprinting of Substance P at the Air- Water Interface
The FIDI spectrum of substance P (SP) after a 30 s exposure to the DBDS is shown in Figure 5.2. No sodium salts were introduced during sample preparation, yet sodium is found to be a common contaminant in mass spectrometry240 and is observed in the mass spectrum of SP in the form of positively charged adducts with the peptide. Intensities of the parent and products are found as mixtures of protonated and sodiated adducts, forming doubly and triply charged ions at m/z 674 ([SPH2]2+), 685 ([SPH + Na]2+), 450 ([SPH3]3+), and 457 ([SPH2 + Na]3+). Singly charged SP species were not present. Over the OH exposure, products
incorporating a single oxygen atom are found at m/z 282 and 455, for the doubly protonated and the triply protonated species, respectively. Singly oxygenated substance P is also detected in the form of the charged sodium adducts at m/z 693 ([SPH + Na + O]2+) and 463 ([SPH2 + Na + O]3+). In addition, appreciable quantities of more highly oxidized compounds are found as doubly and triply oxygenated species at m/z 690 and 698, respectively, both doubly protonated. The 16 Da mass shift is indicative that oxygen incorporation has occurred, yet is inconclusive as to which residue is the primary target of the OH radical. The hydrophobic C-terminus of SP contains phenylalanine, leucine, and methionine, all of which can produce an SP + 16 Da product based on Scheme 5.1a.
Figure 5.2. Positive mode FIDI mass spectrum of the surface of a 150 μM droplet of substance P (SP) after a 30 s exposure to the DBDS. N = 3.
The singly oxidized m/z 282 species was isolated and subjected to FIDI-MS/MS analysis.
The resulting collision-induced dissociation (CID) spectrum of this species under the same oxidative conditions is found in Figure 5.3. The most abundant fragment is found at m/z
650.42, a loss which, when taking into account that the ions are doubly charged, corresponds to a mass of 64 Da. This loss is well-documented in the fragmentation of oxidized methionine residues in proteomic studies as the neutral methane sulfenic acid (CH3SOH).241-244 The pathway for this loss is shown in Scheme 5.2. The sulfoxide oxygen deprotonates a neighboring methylene unit, enabling the formation of the carbon-carbon double bond while displacing neutral methanesulfenic acid (CH3SOH). The large abundance of this species confirms that the methionine residue is the primary site of oxidation in SP.
Furthermore, the presence of b-type ions, N-terminal positively charged fragment ions produced by scission of the peptide bond, is found with varying residue numbers.
Importantly, the b10 fragment is observed as both the singly charged and doubly charged species at m/z 1199.58 and 600.33, respectively. The existence of these ions in significant quantities in the mass spectrum reveals that the site of oxidation does not occur appreciably on the ten N-terminal residues of substance P, but rather provides a second source of evidence that the site of oxidation is the methionine residue. Many bn ions reside at a mass-to-charge ratio identical to native SP, indicating that the constituent n residues are not oxidation sites.
The mass shifted [b7 + O]+ and [b8 + O]+ at m/z 899.50 and m/z 1046.50, respectively, suggest a product with oxidation site within the first 7 and 8 residues. With no other evidence that residues 1 – 6 carry the oxidation site, these peaks imply oxidation at residue 7 or 8, both of which are phenylalanine residues. However, the total relative intensity makes up only 10%
of the major peak at m/z 650.42, which is definitive proof of methionine oxidation. This suggests that there is a significant bias toward methionine oxidation rather than phenylalanine oxidation.
Figure 5.3. Collision-induced dissociation (CID) analysis of the singly oxidized substance P at m/z 682. Fragment ions are labeled using b- and y-type ion designation commonly used in proteomics.
Inset: diagram of the locations of the peptide bond breakage along the SP backbone that produces the bn ions found in the spectrum. Hydrophobic residues are highlighted in red. N = 3.
Scheme 5.2. Proposed mechanism of the formation of m/z 650 in the collision-induced dissociation of singly oxidized substance P ([SPH2 + O]2+) during FIDI-MS/MS analysis by the loss of methanesulfenic acid (CH3SOH) from oxidized methionine.
The results of Figure 5.3 implicate the air-water interface as an environment that influences peptide reactivity with the hydroxyl radical to a considerable degree. Aqueous phase reaction rates predict that the methionine, phenylalanine, and arginine are the most susceptible to OH-mediated oxidation, with reaction rates within an order of magnitude of
each other (8.5 × 109, 6.9 × 109, 3.5 × 109 M–1 s–1, respectively).218, 245 However, the CID spectrum indicates that the oxidation of the phenylalanine residue constitutes a minor pathway relative to the oxidation of the methionine residue. The striking difference in relative amounts of phenylalanine versus methionine oxidation product despite their similar kinetics suggest the experiment detects differences in accessibility of the two residue types to the gas phase OH. Furthermore, the absence of the characteristic SP – 43 Da species caused by arginine oxidation is also indicative of residue accessibility to the gas phase, as arginine is found on as the N-terminus. The results suggest that the OH does not appreciably penetrate the surface of the SP monolayer in order to oxidize this and other residues in this hydrophilic region.