Oxidative modifications to the side chains of sulfur-containing amino acids often limit the number of product ions formed during collision-induced dissociation (CID) and thus make it difficult to obtain sequence information for oxidized peptides. In this work, we demonstrate that electron-transfer dissociation (ETD) can be used to improve the sequence information obtained from peptides with oxidized cysteine and methionine residues. In contrast to CID, ETD is found to be much less sensitive to the side-chain chemistry, enabling extensive sequence information to be obtained in cases where CID fails to provide this information. These results indicate that ETD is a valuable technique for studying oxidatively modified peptides and proteins. In addition, we report a unique and very abundant product ion that is formed in the CID spectra of peptides having N-terminal cysteine sulfinic acid residues. The mechanism for this unique dissociation pathway involves a six-membered cyclic intermediate and leads to the facile loss of NH 3 and SO 2 , which corresponds to a mass loss of 81 Da. While the facile nature of this dissociation pathway limits the sequence information present in CID spectra of peptides with N-terminal cysteine sulfinic acid residues, extensive sequence information for these peptides can be obtained with ETD. M ass spectrometry (MS) is widely used for sequencing and identifying amino acid modifications in peptides and proteins. Identifying modifications to proteins is important for a variety of reasons. Post-translational modifications (PTMs) of proteins are necessary for a wide range of cellular functions such as protein trafficking, protein-protein interactions, and transcription. Identifying and pinpointing these modification sites are important for more deeply understanding protein function, both normal and abnormal. In this context, PTMs such as phosphorylation, acetylation, glycosylation, sulfonation, and methylation are important to characterize. Oxidation is another important protein modification that is typically associated with oxidative stress [1-4], but recent work has also shown that protein oxidation can play a regulatory role as well [5]. Furthermore, an increasing number of techniques make use of oxidative labeling to study protein structure. These methods use radicals (e.g., · OH) to modify solvent-exposed [6 -9] or metal-bound amino acids [10 -17], and MS n to identify oxidatively modified residues, typically in conjunction with proteolytic digestion.Very often side-chain modifications to peptides can make sequencing by collision-induced dissociation (CID) difficult. Perhaps the most well known example is the effect of phosphorylation on peptide ion dissociation. The CID spectra of phosphorylated peptides are commonly dominated by a neutral loss of H 3 PO 4 , often with little other sequence information present. Similarly, side-chain oxidation can dramatically affect peptide dissociation patterns and limit sequence information that is available by CID. For example, oxidation of cysteine ...

While the Cu(II) binding sites of the prion protein have been well studied under Cu-saturation conditions, the identity of the residues involved in coordinating Cu(II) at low stoichiometries and the order in which the binding sites load with Cu(II), remain unresolved. In this study, we have used two mass spectrometry based methods to gather insight into Cu(II)-prion binding under different stoichiometric loadings of Cu(II). The first method uses metal-catalyzed oxidation reactions to site specifically modify the residues bound to Cu(II) in solution, and the second method determines Cu binding sites based on the protection of His from modification by diethyl pyrocarbonate when this residue binds Cu(II) in solution. For both methods, the residues that are labeled by these reactions can then be unambiguously identified using tandem mass spectrometry. Upon applying these two complementary methods to a construct of the prion protein that contains residues 23-28 and 57-

A multiplexing method for performing MS/MS on multiple peptide ions simultaneously in a quadrupole ion trap mass spectrometer (QITMS) has been developed. This method takes advantage of the inherent mass bias associated with ion accumulation in the QITMS to encode the intensity of precursor ions in a way that allows the corresponding product ions to be identified. The intensity encoding scheme utilizes the Gaussian distributions that characterize the relationship between ion intensities and rf trapping voltages during ion accumulation. This straightforward approach uses only two arbitrary waveforms, one for isolation and one for dissociation, to gather product ion spectra from N precursor ions in as little as two product ion spectra. In the example used to illustrate this method, 66% of the product ions from five different precursor peptide ions were correctly correlated using the multiplexing approach. Of the remaining 34% of the product ions, only 6% were misidentified, while 28% of the product ions failed to be identified because either they had too low intensity or they had the same m/z ratio as one of the precursor ions or the same m/z ratio as a product ion from a different precursor ion. This method has the potential to increase sample throughput, reduce total analysis times, and increase signal-to-noise ratios as compared to conventional MS/MS methods.