Specific detection of protein carbonylation sites by 473 nm photodissociation mass spectrometry.

The study of protein oxidation remains a challenge despite the biomedical interest in reliable biomarkers of oxidative stress. This is particularly true for carbonylations although, recently, liquid chromatography-mass spectrometry techniques (LC-MS) have been proposed to detect this non-enzymatic and poorly distributed oxidative modification of proteins using untargeted or carbonyl-reactive probe methods. These methods proved to be feasible but could not preserve the dynamic range of the protein sample, making it impossible to quantify oxidatively modified proteoforms compared with native proteoforms. Here, we propose an innovative method based on the implementation of a reactive carbonyl probe conjugated with a laser-sensitive chromophore, dabcyl-aminooxy, which confers optical specificity to the LC-MS approach. In addition, our protein carbonyl detection method allows us to localize individual carbonylation sites by observing fragments of derivatized oxidized peptides. Two model proteins, alpha-synuclein and beta-lactoglobulin, were oxidized and carbonylation sites were detected, resulting in the identification of respectively 34 and 77 different carbonylated amino acids. Thus, we demonstrated the application of a direct and sensitive method for studying protein carbonylation sites in complex protein extracts.

Structural and dynamical insights into SilE silver binding from combined analytical probes.

Silver has been used for its antimicrobial properties to fight infection for thousands of years. Unfortunately, some Gram-negative bacteria have developed silver resistance causing the death of patients in a burn unit. The genes responsible for silver resistance have been designated as the sil operon. Among the proteins of the sil operon, SilE has been shown to play a key role in bacterial silver resistance. Based on the limited information available, it has been depicted as an intrinsically disordered protein that folds into helices upon silver ion binding. Herein, this work demonstrates that SilE is composed of 4 clearly identified helical segments in the presence of several silver ions. The combination of analytical and biophysical techniques (NMR spectroscopy, CD, SAXS, HRMS, CE-ICP-MS, and IM-MS) reveals that SilE harbors four strong silver binding sites among the eight sites available. We have also further evidenced that SilE does not adopt a globular structure but rather samples a large conformational space from elongated to more compact structures. This particular structural organization facilitates silver binding through much higher accessibility of the involved His and Met residues. These valuable results will advance our current understanding of the role of SilE in the silver efflux pump complex mechanism and will help in the future rational design of inhibitors to fight bacterial silver resistance.

Unbiased Detection of Cysteine Sulfenic Acid by 473 nm Photodissociation Mass Spectrometry: Toward Facile In Vivo Oxidative Status of Plasma Proteins.

Cysteine (Cys) is prone to diverse post-translational modifications in proteins, including oxidation into sulfenic acid (Cys-SOH) by reactive oxygen species generated under oxidative stress. Detection of low-concentration and metastable Cys-SOH within complex biological matrices is challenging due to the dynamic concentration range of proteins in the samples. Herein, visible laser-induced dissociation (LID) implemented in a mass spectrometer was used for streamlining the detection of Cys oxidized proteins owing to proper derivatization of Cys-SOH with a chromophore tag functionalized with a cyclohexanedione group. Once grafted, peptides undergo a high fragmentation yield under LID, leading concomitantly to informative backbone ions and to a chromophore reporter ion. Seventy-nine percent of the Cys-containing tryptic peptides derived from human serum albumin and serotransferrin tracked by parallel reaction monitoring (PRM) were detected as targets subjected to oxidation. These candidates as well as Cys-containing peptides predicted by in silico trypsin digestion of five other human plasma proteins were then tracked in real plasma samples to pinpoint the endogenous Cys-SOH subpopulation. Most of the targeted peptides were detected in all plasma samples by LID-PRM, with significant differences in their relative amounts. By eliminating the signal of interfering co-eluted compounds, LID-PRM surpasses conventional HCD (higher-energy collisional dissociation)-PRM in detecting grafted Cys-SOH-containing peptides and allows now to foresee clinical applications in large human cohorts.

Ionization and Photofragmentation of Isolated Metalloporphyrin Cations Investigated by VUV Action Spectroscopy*.

We investigated the photoionization and fragmentation of isolated metal protoporphyrin IX cations (MPPIX+ with M=Fe, Co, Zn) by means of vacuum-ultraviolet (VUV) action spectroscopy in the energy range of 8.5-35 eV. Experiments were carried out in the gas phase by interfacing an electrospray ionization tandem mass spectrometer with a synchrotron beamline. The mass spectra and partial ion yields show that photoexcitation of the precursor ions predominantly leads to . CH2 COOH radical side-chain losses of the macrocycle with additional methyl radical (. CH3 ) side-chain losses. Ionization, in contrast, leads to the formation of the intact ionized precursor and various doubly charged fragments which are mostly due to side-chain cleavages. Although statistical fragmentation dominates, we found evidence for non-statistical processes such as new fragments involving for example single and double H2 O losses, indicating that different relaxation mechanisms are at play upon photoionization compared to photoexcitation. The measured ionization energies were 9.6±0.2 eV, 9.4±0.2 eV and 9.6±0.2 eV for FePPIX+ , CoPPIX+ and ZnPPIX+ , respectively.

Increasing specificity of tandem mass spectrometry by laser-induced dissociation.

Mass spectrometry offers an arsenal of tools for diverse proteomic investigations. This perspective article reviews some of the recent developments in the field of coupling laser-induced dissociation with mass spectrometry (LID-MS). Strategies involving labelling with a chromophore to induce specific photo-absorption properties are considered, with a focus on specific amino acid derivatization. Some of the opportunities and challenges of LID-MS after targeted labelling for increasing specificity in complex sample analysis are discussed.

Data-Independent Acquisition Coupled to Visible Laser-Induced Dissociation at 473 nm (DIA-LID) for Peptide-Centric Specific Analysis of Cysteine-Containing Peptide Subset.

Thanks to comprehensive and unbiased sampling of all precursor ions, the interest to move toward bottom-up proteomic with data-independent acquisition (DIA) is continuously growing. DIA offers precision and reproducibility performances comparable to true targeted methods but has the advantage of enabling retrospective data testing with the hypothetical presence of new proteins of interest. Nonetheless, the chimeric nature of DIA MS/MS spectra inherent to concomitant transmission of a multiplicity of precursor ions makes the confident identification of peptides often challenging, even with spectral library-based extraction strategy. The introduction of specificity at the fragmentation step upon ultraviolet or visible laser-induced dissociation (LID) range targeting only the subset of cysteine-containing peptides (Cys-peptide) has been proposed as an option to streamline and reduce the search space. Here, we describe the first coupling between DIA and visible LID at 473 nm to test for the presence of Cys-peptides with a peptide-centric approach. As a test run, a spectral library was built for a pool of Cys-synthetic peptides used as surrogates of human kinases (1 peptide per protein). By extracting ion chromatograms of query standard and kinase peptides spiked at different concentration levels in an Escherichia coli proteome lysate, DIA-LID demonstrates a dynamic range of detection of at least 3 decades and coefficients of precision better than 20%. Finally, the spectral library was used to search for endogenous kinases in human cellular extract.

Think Negative: Finding the Best Electrospray Ionization/MS Mode for Your Analyte.

For the first time, the electrospray ionization efficiency (IE) scales in positive and negative mode are united into a single system enabling direct comparison of IE values across ionization modes. This is made possible by the use of a reference compound that ionizes to a similar extent in both positive and negative modes. Thus, choosing the optimal (i.e., most sensitive) ionization conditions for a given set of analytes is enabled. Ionization efficiencies of 33 compounds ionizing in both modes demonstrate that, contrary to general practice, negative mode allows better sensitivity for 46% of such compounds whereas the positive mode is preferred for only 18%, and for 36%, the results for both modes are comparable.

Fragmentation patterns of chromophore-tagged peptides in visible laser induced dissociation.

RATIONALE: Tandem mass spectrometry (MS/MS) is the pivotal tool for protein structural characterization and quantification. Identification relies on the fragmentation step of tryptic peptides in bottom-up strategy. Specificity of fragmentation can be obtained using laser-induced dissociation (LID) in the visible range, after tagging of the targeted peptides with an adequate chromophore. Backbone fragmentation is required to obtain specific fragments and confident identification. We present herein a study of fragmentation patterns of chromophore-tagged peptides in LID, showing the potential of LID methodology to provide the maximum number of fragments for further identification and quantification. METHODS: A total of 401 cysteine-containing tryptic peptides originating from the human proteome were derivatizated on the thiol group of cysteine with a Dabcyl maleimide chromophore, which has a high photo-absorption cross section at 473 nm. The derivatized peptides were then analyzed by LID at 473 nm on a Q Exactive instrument. RESULTS: LID spectra present a characteristic fragment at m/z 252.112 for all precursors. This product ion arises from the internal dissociation of the Dabcyl chromophore. Several peptide-backbone fragment ions are also detected. Results show the quasi absence of fragmentation at the cysteine site. This indicates that part of the energy must be redistributed across the entire system despite excitation initially localized at the chromophore. Indeed, the fragmentation mainly occurs at 3 to 5 amino acids from the derivatized cysteine residue. CONCLUSIONS: LID of derivatized cysteine-containing peptides displays the initial fragmentation of the chromophore. As energy is redistributed all along the peptide sequence, fragmentation of the peptide backbone is also observed. Thus, LID of chromophore-tagged peptides produces adequate fragment ions, allowing both good sequence coverage for a greater confidence of identification, and a large choice of transitions for specific quantification.

Temperature Response of Rhodamine B-Doped Latex Particles. From Solution to Single Particles.

Nanoparticle-based temperature imaging is an emerging field of advanced applications. Herein, the sensitivity of the fluorescence of rhodamine B-doped latex nanoparticles toward temperature is described. Submicrometer size latex particles were prepared by a surfactant-free emulsion polymerization method that allowed a simple and inexpensive way to incorporate rhodamine B into the nanoparticles. Also, rhodamine B-coated latex nanoparticles dispersed in water were prepared in order to address the effect of the dye location in the nanoparticles on their temperature dependence. A better linearity of the temperature dependence emission of the rhodamine B-embedded latex particles, as compared to that of free rhodamine B dyes or rhodamine B-coated latex particles, is observed. Temperature-dependent fluorescence measurements by fluorescent confocal microscopy on individual rhodamine B-embedded latex particles were found similar to those obtained for fluorescent latex nanoparticles in solution, indicating that these nanoparticles could be good candidates to probe thermal processes as nanothermometers.

Correlating droplet size with temperature changes in electrospray source by optical methods.

We investigated how the temperature and size of charged droplets are affected by the electrospray ionization (ESI) process, using in situ measurements involving laser-induced fluorescence and Mie scattering on a thermal gradient focusing ESI source. Rhodamine dyes were employed as temperature indicators using ratiometric intensity-based fluorescence techniques. The results were compared to lifetime-based techniques using tris(2,2'-bipyridyl)dichlororuthenium(II) hexahydrate, [Ru(bpy)3](2+). Both methods gave similar profiles. Nevertheless, the precision and sensitivity were higher for lifetime-based techniques in comparison to intensity-based techniques. Global warming (with ΔT ∼10 K) of the ESI plume is reported while the size of the droplet decreases along the plume. The global warming indicates that the conductive thermal transfer (between the superheated sheath gas and the solvent) is predominant and stronger than the cooling effect due to the evaporation of the droplets, and this outcome is effectively reproduced by a diffusion-controlled evaporation model. Thermal gradient focusing ESI sources therefore appear to be efficient sources for evaporating large amounts of solvent, along with an increase in temperature.

Formation and characterisation of the silver hydride nanocluster cation [Ag3H2((Ph2 P)2CH2)](+) and its release of hydrogen.

Multistage mass spectrometry and density functional theory (DFT) were used to characterise the small silver hydride nanocluster, [Ag3 H2 L](+) (where L=(Ph2 P)2 CH2 ) and its gas-phase unimolecular chemistry. Collision-induced dissociation (CID) yields [Ag2 HL](+) as the major product while laser-induced dissociation (LID) proceeds via H2 formation and subsequent release from [Ag3 H2 L](+) , giving rise to [Ag3 L](+) as the major product. Deuterium labelling studies on [Ag3 D2 L](+) prove that the source of H2 is from the hydrides and not from the ligand. Comparison of TD-DFT absorption patterns obtained for the optimised structures with action spectroscopy results, allows assignment of the measured features to structures of precursors and products. Molecular dynamics "on the fly" reveal that AgH loss is favoured in the ground state, but H2 formation and loss is preferred in the first excited state S1 , in agreement with CID and LID experimental findings. This indicates favourable photo-induced formation of H2 and subsequent release from [Ag3 H2 L](+) , an important finding in context of metal hydrides as a hydrogen storage medium, which can subsequently be released by heating or irradiation with light.

Imaging of whole zebra fish (Danio rerio) by desorption electrospray ionization mass spectrometry.

RATIONALE: To demonstrate the potential use of zebra fish (Danio rerio) as a model vertebrate organism by producing two-dimensional ion images of the whole zebra fish, and being able to distinguish particular areas of interest such as the brain, spinal cord, and stomach region using a desorption electrospray ionization (DESI) ion source coupled to a linear ion trap. METHODS: Imaging experiments are performed on 45 µm sagittal slices of zebra fish (Danio rerio), which are thaw-mounted onto microscope glass slides. The slides are then analyzed using a solvent of acetonitrile/dimethylformamide (50:50) (ACN/DMF), with a solvent flow rate of 1.5 µL/min; data are acquired in negative ion mode. Raw mass spectrum data files are converted into a readable file for Biomap. The images produced are then analyzed for ion distributions. RESULTS: We are able to create clear, distinct, chemical intensity images of the brain, spinal cord, and stomach based on lipid content as well as bile salt. The identities of these compounds were confirmed by tandem mass spectrometric (MS/MS) experiments and comparisons with literature. CONCLUSIONS: Imaging of whole zebra fish is possible using ambient ionization techniques such as DESI. Analyses are fast and reliable. For most of the compounds observed, the identification by MS/MS can be performed directly from the fish tissue sample.

Implementing visible 473 nm photodissociation in a Q-Exactive mass spectrometer: towards specific detection of cysteine-containing peptides.

Improvement of the fragmentation specificity may streamline data processing of bottom-up proteomic experiments by drastically reducing either the amount of MS/MS data to process in the discovery phase or the detection of interfering signals in targeted quantification. Photodissociation at appropriate wavelengths is a promising alternative technique to the non-discriminating conventional activation mode by collision. Here, we describe the implementation of visible LID at 473 nm in a Q-Exactive-Orbitrap mass spectrometer for the specific detection of cysteine-containing peptides tagged with a Dabcyl group. HCD cell DC offset and irradiation time were optimized to obtain high fragmentation yield and spectra free of contaminating CID product ions, while keeping the irradiation time scale compatible with chromatographic separation. With this optimized experimental set-up, the selective detection of cysteine-containing peptides in a whole tryptic hydrolysate of three combined proteins is demonstrated by comparing all ion fragmentation (AIF) spectra recorded online with and without laser irradiation.

Total ApoE and ApoE4 isoform assays in an Alzheimer's disease case-control study by targeted mass spectrometry (n=669): a pilot assay for methionine-containing proteotypic peptides.

Allelic polymorphism of the apolipoprotein E (ApoE) gene (ApoE ε2, ApoE ε3 and ApoE ε4 alleles) gives rise to three protein isoforms (ApoE2, ApoE3 and ApoE4) that differ by 1 or 2 amino acids. Inheritance of the ApoE ε4 allele is a risk factor for developing Alzheimer's disease (AD). The potential diagnostic value of ApoE protein levels in biological fluids (i.e. cerebrospinal fluid, plasma and serum) for distinguishing between AD patients and healthy elderly subjects is subject to great controversy. Although a recent study reported subnormal total ApoE and ApoE4 levels in the plasma of AD patients, other studies have found normal or even elevated protein levels (versus controls). Because all previously reported assays were based on immunoenzymatic techniques, we decided to develop an orthogonal assay based on targeted mass spectrometry by tracking (i) a proteotypic peptide common to all ApoE isoforms and (ii) a peptide that is specific for the ε4 allele. After trypsin digestion, the ApoE4-specific peptide contains an oxidation-prone methionine residue. The endogenous methionine oxidation level was evaluated in a small cohort (n=68) of heterozygous ε3ε4 carriers containing both healthy controls and AD patients. As expected, the proportion of oxidized residues varied from 0 to 10%, with an average of 5%. We therefore developed a standardized strategy for the unbiased, absolute quantification of ApoE4, based on performic acid oxidization of methionine. Once the sample workflow had been thoroughly validated, it was applied to the concomitant quantification of total ApoE and ApoE4 isoform in a large case-control study (n=669). The final measurements were consistent with most previously reported ApoE concentration values and confirm the influence of the different alleles on the protein expression level. Our results illustrate (i) the reliability of selected reaction monitoring-based assays and (ii) the value of the oxidization step for unbiased monitoring of methionine-containing proteotypic peptides. Furthermore, a statistical analysis indicated that neither total ApoE and ApoE4 levels nor the ApoE/ApoE4 ratio correlated with the diagnosis of AD. These findings reinforce the conclusions of previous studies in which plasma ApoE levels had no obvious clinical significance.

Characterization of Phosphorylated Peptides by Electron-Activated and Ultraviolet Dissociation Mass Spectrometry: A Comparative Study with Collision-Induced Dissociation.

Mass-spectrometry-based methods have made significant progress in the characterization of post-translational modifications (PTMs) in peptides and proteins; however, room remains to improve fragmentation methods. Ideal MS/MS methods are expected to simultaneously provide extensive sequence information and localization of PTM sites and retain labile PTM groups. This collection of criteria is difficult to meet, and the various activation methods available today offer different capabilities. In order to examine the specific case of phosphorylation on peptides, we investigate electron transfer dissociation (ETD), electron-activated dissociation (EAD), and 193 nm ultraviolet photodissociation (UVPD) and compare all three methods with classical collision-induced dissociation (CID). EAD and UVPD show extensive backbone fragmentation, comparable in scope to that of CID. These methods provide diverse backbone fragmentation, producing a/x, b/y, and c/z ions with substantial sequence coverages. EAD displays a high retention efficiency of the phosphate modification, attributed to its electron-mediated fragmentation mechanisms, as observed in ETD. UVPD offers reasonable retention efficiency, also allowing localization of the PTM site. EAD experiments were also performed in an LC-MS/MS workflow by analyzing phosphopeptides spiked in human plasma, and spectra allow accurate identification of the modified sites and discrimination of isomers. Based on the overall performance, EAD and 193 nm UVPD offer alternative options to CID and ETD for phosphoproteomics.

Improved detection specificity for plasma proteins by targeting cysteine-containing peptides with photo-SRM.

Targeted mass spectrometry using selected reaction monitoring (SRM) has emerged as an alternative to immunoassays for protein quantification owing to faster development time and higher multiplexing capability. However, the SRM strategy is faced with the high complexity of peptide mixtures after trypsin digestion of whole plasma or the cellular proteome that most of the time causes contamination, irremediably, by interfering compounds in the transition channels monitored. This problem becomes increasingly acute when the targeted protein is present at a low concentration. In this work, the merit of laser-induced photo-dissociation in the visible region at 473 nm implemented in an hybrid quadrupole linear ion-trap mass spectrometer (photo-SRM) was evaluated for detection specificity of cysteine-containing peptides in a group of plasma proteins after tagging with a dabcyl chromophore. Compared with conventional SRM, photo-SRM chromatograms have improved detection specificity for most of peptides monitored. Comparison of the signals obtained for the best proteotypic peptides in SRM mode and those recorded by photo-SRM of cysteine-containing peptides for the same proteins reveals either increased (up to 10-fold) or similar signal to photo-SRM detection. Finally, photo-SRM has extended response linearity across a calibration plot obtained by diluting human plasma in rat plasma, down to the lowest concentrations. Hence, photo-SRM may advantageously complement conventional SRM in assay of proteins in complex biological matrices.

Profiling an electrospray plume by laser-induced fluorescence and Fraunhofer diffraction combined to mass spectrometry: influence of size and composition of droplets on charge-state distributions of electrosprayed proteins.

We investigated how physico-chemical properties of charged droplets are affected by the electrospray process, using simultaneous in situ measurements by laser-induced fluorescence (LIF), Fraunhofer diffraction and mass spectrometry. For this purpose, we implemented a laser-induced-fluorescence profiling setup in conjunction with a fast, high-resolution particle sizing scheme on a modified Agilent Jet Stream electrospray source coupled to a single quadrupole mass analyser. The optical setup permits us to profile the solvent fractionation and the size of the droplets as they evaporate in an electrospray plume by measuring both the angular scattering pattern and emission spectra of a solvatochromic fluorescent dye. Mass spectra are recorded simultaneously. These mass spectrometry and optical spectroscopy investigations allow us to study the relation between the observed charge-state distributions of protein anions and physico-chemical properties of evaporating droplets in the spray plume. By mixing water with methanol, a refolding of cytochrome C is observed as the water percentage increases in the plume due to the preponderant evaporation of volatile methanol.

Relative quantification of sulfenic acids in plasma proteins using differential labelling and mass spectrometry coupled with 473 nm photo-dissociation analysis: A multiplexed approach applied to an Alzheimer's disease cohort.

Cysteine (Cys) is subject to a variety of reversible post-translational modifications such as formation of sulfenic acid (Cys-SOH). If this modification is often involved in normal biological activities, it can also be the result of oxidative damage. Indeed, oxidative stress yields abnormal cysteine oxidations that affect protein function and structure and can lead to neurodegenerative diseases. In a context of population ageing, validation of novel biomarkers for detection of neurodegenerative diseases is important. However, Cys-SOH proteins investigation in large human cohorts is challenging due to their low abundance and lability under endogenous conditions. To improve the detection specificity towards the oxidized protein subpopulation, we developed a method that makes use of a mass spectrometer coupled with visible laser induced dissociation (LID) to add a stringent optical specificity to the mass selectivity. Since peptides do not naturally absorb in the visible range, this approach relies on the proper chemical derivatization of Cys-SOH with a chromophore functionalized with a cyclohexanedione. To compensate for the significant variability in total protein expression within the samples and any experimental bias, a normalizing strategy using free thiol (Cys-SH) cysteine peptides derivatized with a maleimide chromophore as internal references was used. Thanks to the differential tagging, oxidative ratios were then obtained for 69 Cys-containing peptides from 19 proteins tracked by parallel reaction monitoring (PRM) LID, in a cohort of 49 human plasma samples from Alzheimer disease (AD) patients. A statistical analysis indicated that, for the proteins monitored, the Cys oxidative ratio does not correlate with the diagnosis of AD. Nevertheless, the PRM-LID method allows the unbiased, sensitive and robust relative quantification of Cys oxidation within cohorts of samples.

Mapping lipid alterations in traumatically injured rat spinal cord by desorption electrospray ionization imaging mass spectrometry.

Desorption electrospray ionization (DESI) mass spectrometry (MS) is used in an imaging mode to interrogate the lipid profiles of 15 µm thin tissue cross sections of injured rat spinal cord and normal healthy tissue. Increased relative intensities of fatty acids, diacylglycerols, and lysolipids (between +120% and +240%) as well as a small decrease in intensities of lipids (-30%) were visualized in the lesion epicenter and adjacent areas after spinal cord injury. This indicates the hydrolysis of lipids during the demyelination process due to activation of phospholipase A(2) enzyme. In addition, signals corresponding to oxidative degradation products, such as prostaglandin and hydroxyeicosatetraenoic acid, exhibited increased signal intensity by a factor of 2 in the negative ion mode in lesions relative to the normal healthy tissue. Analysis of malondialdehyde, a product of lipid peroxidation and marker of oxidative stress, was accomplished in the ambient environment using reactive DESI mass spectrometry imaging. This was achieved by electrospraying reagent solution containing dinitrophenylhydrazine as high-velocity charged droplets onto the tissue section. The hydrazine reacts selectively and rapidly with the carbonyl groups of malondialdehyde, and signal intensity of twice the intensity was detected in the lesions compared to healthy spinal cord. With a small amount of tissue sample, DESI-MS imaging provides information on the composition and distribution of specific compounds (limited by the occurrence of isomeric lipids with very similar fragmentation patterns) in lesions after spinal cord injury in comparison with normal healthy tissue allowing identification of the extent of the lesion and its repair.

End-group characterization of poly(styrene sulfonate sodium salt) by activated electron photo-detachment dissociation.

Tandem mass spectrometry of poly(styrene sulfonate sodium salt) (PSS) was performed after activated electron photo-detachment dissociation (activated EPD). In this technique, doubly charged PSS oligomers were first produced in negative mode electrospray ionization, then oxidized into radical anions upon electron photo-detachment using a 220 nm laser wavelength, and further activated by collision. In contrast to the collision-induced dissociation (CID) of negatively charged PSS oligomers, which does not provide informative data with regard to the end-groups, activated-EPD is shown here to promote radical-induced dissociation reactions thanks to the oxidation of a sulfonate group upon laser irradiation. Major product ions generated after backbone bond cleavages contained one or the other chain terminations and could be accounted for by two main mechanisms. Moreover, each of the proposed dissociation reactions was shown to generate two distinct fragments, depending on the location of the oxidized monomer near one or the other chain terminal moieties. As a result, a combination of these two fragments allowed a straightforward mass characterization of each end-group.