CID Conditions Research Articles

Structures and unimolecular chemistry of alkali metal cation complexes with glutathione in the gas phase

This study investigates the unimolecular reactions of glutathione complexes with alkali metal cations in the gas phase through sustained off-resonance irradiation collision-induced dissociation and examines their structures using a combination of infrared multiphoton dissociation spectroscopy and computational techniques. Under soft CID conditions, glutathione complexes with charge-dense cations such as Li⁺, Na⁺, and K⁺ show significant fragmentation of glutathione, while complexes with heavier cations, Rb⁺ and Cs⁺, primarily undergo loss of glutathione. This behavior is attributed to the stronger non-covalent binding between smaller metal cations and glutathione, which competes with the dissociation thresholds of covalent interactions within the peptide complex. Using CREST, a tool for determining trial structures which were submitted to density functional theory calculations, a thorough investigation of the conformational space revealed many possible structures, including pentacoordinated structures for the Na⁺ and K⁺ complexes, as well as tetra-tri-, bi-, and monocoordinated structures along with zwitterionic structures for all metal cation/GSH complexes. For all alkali metal cation complexes, the thermodynamically most stable structures were found to be tetracoordinated A-type structures where the metal cation is bound to the amine nitrogen and three of the carbonyl oxygens—all except O2, the amide between glycine and cysteine. These computed infrared spectra for these lowest energy complexes were also consistent with the experimental vibrational spectra in the fingerprint region. Based on relative energies and the comparison of computed and experimental infrared spectra in the fingerprint region, the tetracoordinate A-type structures are concluded to be the dominant forms of the [M(GSH)]+ complexes in the gas phase.

First Direct Evidence of Interpartner Hydride/Deuteride Exchanges for Stored Sodiated Arginine/Fructose-6-phosphate Complex Anions within Salt-Solvated Structures.

Mass spectrometric investigations of noncovalent binding between low molecular weight compounds revealed the existence of gas-phase (GP) noncovalent complex (NCC) ions involving zwitterionic structures. ESI MS is used to prove the formation of stable sodiated NCC anions between fructose (F6P) and arginine (R) moieties. Theoretical calculations indicate a folded solvated salt (i.e., sodiated carboxylate interacting with phosphate) rather than a charge-solvated form. Under standard CID conditions, [(F6P+R-H+Na)-H]- competitively forms two major product ions (PIs) through partner splitting [(R-H+Na) loss] and charge-induced cross-ring cleavage while preserving the noncovalent interactions (noncovalent product ions (NCPIs)). MS/MS experiments combined with in-solution proton/deuteron exchanges (HDXs) demonstrated an unexpected labeling of PIs, i.e., a correlated D-enrichment/D-depletion. An increase in activation time up to 3000 ms favors such processes when limited to two H/D exchanges. These results are rationalized by interpartner hydride/deuteride exchanges (⟨HDX⟩) through stepwise isomerization/dissociation of sodiated NCC-d11 anions. In addition, the D-enrichment/D-depletion discrepancy is further explained by back HDX with residual water in LTQ (selective for the isotopologue NCPIs as shown by PI relaxation experiments). Each isotopologue leads to only one back HDX unlike multiple HDXs generally observed in GP. This behavior shows that NCPIs are zwitterions with charges solvated by a single water molecule, thus generating a back HDX through a relay mechanism, which quenches the charges and prevents further back HDX. By estimating back HDX impact on D-depletion, the interpartner ⟨HDX⟩ complementarity was thus illustrated. This is the first description of interpartner ⟨HDX⟩ and selective back HDX validating salt-solvated structures.

Radical-induced dissociation leading to the loss of CO2 from the oxazolone ring of [b5- H]˙(+) ions.

Macrocyclization is commonly observed in large bn(+) (n≥ 4) ions and as a consequence can lead to incorrect protein identification due to sequence scrambling. In this work, the analogous [b5- H]˙(+) radical cations derived from aliphatic hexapeptides (GA5˙(+)) also showed evidence of macrocyclization under CID conditions. However, the major fragmentation for [b5- H]˙(+) ions is the loss of CO2 and not CO loss, which is commonly observed in closed-shell bn(+) ions. Isotopic labeling using CD3 and (18)O revealed that more than one common structure underwent dissociations. Theoretical studies found that the loss of CO2 is radical-driven and is facilitated by the radical being located at the Cα atom immediately adjacent to the oxazolone ring. Comparable energy barriers against macrocyclization, hydrogen-atom transfer, and fragmentations are found by DFT calculations and the results are consistent with the experimental observations that a variety of dissociation products are observed in the CID spectra.

Unexpected formation of [M]2+ from [M+CuCl+H]2+ ions under CID conditions, where M is a molecule of 3,5-bis(2,2’-bipyridin-4-ylethynyl)benzoic acid or its methyl ester

Abstract [M+CuCl+H]2+ ions were generated using electrospray ionization (ESI); where M is a molecule of 3,5-bis(2,2’-bipyridin-4-ylethynyl)benzoic acid or its methyl ester (1 and 2, respectively). The ions were subjected to CID-MS/MS analysis. It was found that their gas phase decomposition lead to the formation of rare di-cations [M]2+, namely [1]2+ and [2]2+ ions. The formation of [1]2+ ion from [3+H+CuCl]2+ ion in the second fragmentation, where 3 is ethyl ester of 3,5-bis(2,2-bipyridin-4-ylethynyl)benzoic acid, was also observed since in the first fragmentation step the loss of ethylene molecule from [3+H+CuCl]2+ ion took place. To the best of our knowledge, it is the first time that [M]2+ ions formation from respective metal complexes has been reported. It is also unusual that formation of [M]2+ ions is not accompanied by formation of [M]+∙ ions. Furthermore, as expected, theoretical calculation and electron ionization mass spectra show that 1 and 2 are not especially prone to form [M]2+ ions. Thus formation of [M]2+ ions under CID conditions is very surprising.

Rearrangement chemistry of an ions probed by IR spectroscopy

The structure and the dynamics of the isomerization of an ions, which are observed upon low-energy collision induced dissociation of protonated peptides in tandem mass spectrometry (MS/MS), are investigated using a combination of gas phase infrared spectroscopy and theory. IR spectra in the fingerprint region are discussed, but a particular emphasis is given to the NH stretching region which turns out to be highly structurally diagnostic. Overall, theory and infrared spectroscopy provide compelling evidence that an ions undergo cyclisation and/or rearrangement reactions. In the cases of the a2 and a3 ions of oligoglycine, the analysis of the NH stretching region is fully consistent with our previous conclusions based on the IR fingerprint spectra. In the case of the a4 ions of oligoglycine, a band observed near 3550cm−1 provides a clear-cut signature of the permuted imine–amide structure, thus allowing for a better structural assignment. The dynamics of the rearrangement chemistry of the imine–amide structure is discussed in details, and a critical discussion on the influence of the experimental CID conditions is proposed in the case of the YG a2 ion.

MS/MS Fragmentation Behavior Study of meso-Phenylporphyrinoids Containing Nonpyrrolic Heterocycles and meso-Thienyl-Substituted Porphyrins

Free base and cobalt(II) complexes of six meso-tetraphenylporphyrinoids containing nonpyrrolic heterocycles and of three meso-thienylporphyrins were investigated using electrospray ionization tandem mass spectrometry (ESI-MS/MS). Their fragmentation was studied in a quadrupole ion trap as a function of the porphyrinoid macrocycle structure and compared with the fragmentation behavior of the benchmark compound meso-tetraphenylporphyrin. In situ oxidation of the neutral cobalt(II) complexes under ESI conditions produced singly charged cobalt(III) porphyrinoid ions; the free bases were ionized by protonation. For the porphyrinoids with an intact porphyrin core, the major fragmentation pathways observed were the losses of the meso-substituent (for meso-phenyl groups) and characteristic fragmentations of one or more meso-substituents (for the meso-thienyl group). Complex fragmentation pathways were observed for porphyrinoids with modifications to the porphyrin core but chemically reasonable structures could be assigned to most fragments, thus delineating general patterns for the behavior of pyrrole-modified porphyrins under CID conditions. ᅟ

ESI formation of a Meisenheimer complex from tetryl and its unusual dissociation

The reactivity of the explosive tetryl (N-methyl-N,2,4,6-tetranitroaniline; Mw = 287 u) was studied using electrospray ionization in negative mode. The main species detected in the spectrum corresponds to the ion observed at m/z 318 (previously assumed to be the odd-electron ion [tetryl + HNO](-•), C7H6O9N6). In this study, we show using D-labeling combined with high-resolution mass spectrometry that this species corresponds to an even-electron anion (i.e. C8H8O9N5), resulting from the formation of a Meisenheimer complex between tetryl and the methanol used as the solvent. Fragmentation of this complex under CID conditions revealed an unexpected fragment: the formation of a 2,4,6-trinitrophenoxide anion at m/z 228. (18)O-labeling combined with quantum chemical calculations helped us better understand the reaction pathways and mechanisms involved in the formation of this product ion. This occurs via a transition state leading to a SN2-type reaction, consequently evolving toward an ion-dipole complex. The latter finally dissociates into deprotonated picric acid.

Differential (14)N/(15)N-Labeling of Peptides Using N-Terminal Charge Derivatization with a High-Proton Affinity for Straightforward de novo Peptide Sequencing.

While de novo peptide sequencing is essential in many situations, it remains a difficult task. This is because peptide fragmentation results in complicated and often incomplete product ion spectra. In a previous study, we demonstrated that N-terminal charge derivatization with 4-amidinobenzoic acid (Aba) resulted in improved peptide fragmentation under low-energy CID conditions. However, even with this derivatization, some ambiguity exists, due to difficulties in discriminating between N- and C-terminal fragments. In this study, to specifically identify b-ions from complex product ion spectra, the differential (14)N/(15)N-labeling of peptides was performed using Aba derivatization. (15)N-Labeled Aba was synthesized in the form of a succinimide ester. Peptides were derivatized individually with (14)N-Aba or (15)N-Aba and analyzed by ESI-MS/MS using a linear ion trap-Orbitrap hybrid FTMS system. The N-terminal fragments (i.e., b-ions) were then identified based on m/z differences arising from isotope labeling. By comparing the spectra between (14)N- and (15)N-Aba derivatized peptides, b-ions could be successfully identified based on the m/z shifts, which provided reliable sequencing results for all of the peptides examined in this study. The method developed in this study allows the easy and reliable de novo sequencing of peptides, which is useful in peptidomics and proteomics studies.

Use of liquid chromatography/electrospray ionization tandem mass spectrometry to study the degradation pathways of terbuthylazine (TER) by Typha latifolia in constructed wetlands: identification of a new TER metabolite

S-Triazines are used worldwide as herbicides for agricultural and non-agricultural purposes. Although terbuthylazine (TER) is the second most frequently used S-triazine, there is limited information on its metabolism. For this reason, an analytical method based on liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI MS/MS) has been developed aiming at the identification of TER and its five major metabolites (desisopropyl-hydroxy-atrazine, desethyl-hydroxy-terbuthylazine, desisopropyl-atrazine, hydroxy-terbuthylazine and desethyl-terbuthylazine) in constructed wetland water samples. The separation of TER and its major metabolites was performed by reversed-phase high-performance liquid chromatography (HPLC) on a C(8) column using a gradient elution of aqueous acetic acid 1% (solvent A) and acetonitrile (solvent B), followed by MS/MS analysis on a triple quadrupole mass spectrometer. The data-depended analysis (DDA) scan approach has been employed and the main degradation pathways of both hydroxyl and chloro (dealkylated and alkylated) metabolites are elucidated through the tandem mass spectral (MS/MS) interpretation of triazine fragments under CID conditions. In addition, another major metabolite of TER, namely N2-tert-butyl-N4-ethyl-6-methoxy-1,3,5-triazine-2,4-diamine, has been identified. This methodology can be further employed in biodegradation studies of TER, thus assisting the assessment of its environmental impact.

Direct Evidence for Tautomerization of the Uracil Moiety within the Pb2+/Uridine-5′-monophosphate Complex: A Combined Tandem Mass Spectrometry and IRMPD study

The structure of the [Pb(UMP)-H](+) (UMP = uridine-5'-monophosphate) complex was studied in the gas phase by combining electrospray ionization (ESI), tandem mass spectrometry, and mid-infrared multiple photon dissociation (IRMPD) spectroscopy. The results obtained show that Pb(2+) ions interact not only with the deprotonated phosphate group but also with a carbonyl group of the nucleobase moiety by folding of the mononucleotide, resulting in macrochelates that are not likely to be present in solution. Comparison between the IRMPD and DFT-computed spectra suggests that the ESI-generated complex likely corresponds to a mixture of several structures, and establishes the enolic tautomers as the most abundant species for the [Pb(UMP)-H](+) ion, while the very weak IRMPD signal observed at ∼1763 cm(-1) points to a minor population of oxo forms. Our data also suggest that losing the nucleobase residue under CID conditions does not necessarily mean a lack of interaction between the metal and the nucleobase moiety, as commonly reported in the literature for large oligonucleotides.

Heparin‐like glycosaminoglycan/amine salt‐bridge interactions: A new potential tool for HLGAGs analysis using mass spectrometry

Characterization of glycosaminoglycans poses a challenge for current analytical techniques, as they are highly acidic, polydisperse and heterogeneous compounds. The purpose of this study is the separation and analysis of a partially depolymerized heparin-like glycosaminoglycan by on-line ion-pairing reversed-phase high-performance liquid chromatography/electrospray mass spectrometry. The gas-phase behavior of two synthesized glycosaminoglycans has been investigated. Dibutylamine was found to be the best suited ion-pairing reagents for mass spectrometry analysis. The optimized ion-pairing conditions provide reproducible and easily interpretable electrospray mass spectra in both negative and positive ESI modes. The glycosaminoglycans are detected as a non-covalent complex with amines. In fact, the observed ionic species and their gas-phase dissociation under CID conditions revealed the presence of salt bridge interactions in the gas phase.

Profiling the peptidome of the venom from the social wasp Agelaia pallipes pallipes

The wasp Agelaia pallipes pallipes is one of the most aggressive species from the neotropical region, causing many stinging accidents every year, characterized by severe envenoming reactions. The identification of peptides is important for understanding the envenoming process; however, the tiny amount of venom produced by these insects makes this task a challenge, using classical analytical approaches. Thus, the venom was previously fractionated, and the sequences were obtained through the use of electrospray ionization with a tridimensional ion-trap and time-of-flight mass analysis under CID conditions. This approach permitted the sequence assignment of nine peptides. The presence of type -d and -w ions generated from the fragmentation of the side chains was used to resolve I/L ambiguity. The distinction between K and Q residues was achieved through esterification of the α- and ε-amino groups in the peptides, followed by mass spectrometry analysis. Six of these peptides were short, linear and polycationic, while the three other peptides presented a single disulfide bridge. The use of reduction and alkylation protocols, followed by ESI-IT-TOF/MS analysis under CID conditions, permitted easy sequencing of the three peptides presenting this post-translational modification. These peptides presented activity related to mast cell degranulation, hemolysis, or even the chemotaxis of leukocytes.

ESI Single Quadrupole Mass Spectrometric Investigation of Polyhydroxy Acids

ESI-MS spectra were recorded with a FIA-MS system under CID conditions for 19 polyhydroxy carboxylic acids to assess the analytical selectivity of single quadrupole MS and to evaluate its suitability for the qualitative identification of the target compounds. Suited ESI operation conditions were defined for several sugar acids and their lactones and the pH dependence of the respective acid-lactone conversion was investigated. Besides the common neutral mass losses of H2O and CO2 specific mass losses, presumably belonging to the release of small carbon chain units, were established for isocitric and gluconic acids. A product ion with m/z 75 was part of the MS spectra of all aldonic acids, potentially indicating this structural subgroup of sugar acids. The spectra of other sugar acids and of tartaric, isocitric and shikimic acids had a product ion with m/z 73 in common. The results reveal that the CID mass spectra of several hydroxy acids show characteristic product ion patterns suited for a selective analyte identification.

Dimethyl Isotope Labeling Assisted De Novo Peptide Sequencing

Here, we explore a de novo sequencing strategy in which we combine Lys-N protein digestion with differential isotopic dimethyl labeling to facilitate the (de novo) identification of multiply charged peptides in ESI-MS, both under CID and ETD conditions. For a large fraction of the Lys-N generated peptides, all primary amines are present at the N-terminal lysine, enabling specific labeling of the N-terminus. Differential derivatization of only the peptide N-terminus in combination with the simultaneous fragmentation of the corresponding isotopologues allows the straightforward distinction of N-terminal fragments from C-terminal and internal fragments. Furthermore, also singly and multiply charged N-terminal fragments can easily be distinguished due to the mass differences of the isotope labeled fragment pairs. As a proof of concept, we applied this approach to proteins isolated from an avocado fruit, and were able to partially de novo sequence and correctly align, with green plant homologues, a previously uncharacterized avocado ascorbate peroxidase.

Structure of Pb2+/dCMP and Pb2+/CMP complexes as characterized by tandem mass spectrometry and IRMPD spectroscopy

The structure of Pb2+/cytidine-5′-monophosphate (CMP) and Pb2+/deoxycytidine-5′-monophosphate (dCMP) complexes were probed in the gas phase by combining electrospray ionization, tandem mass spectrometry and mid-infrared multiple-photon dissociation (IRMPD) spectroscopy. The fragment ions detected upon collision suggest that the metal interacts with both the phosphate group and the cytosine. This finding is further confirmed by IRMPD spectroscopy. The IRMPD spectrum of the ESI-generated [Pb(dCMP)−H]+ and [Pb(CMP)−H]+ complexes is indeed in very good agreement with DFT-computed infrared absorption spectra of macrochelate forms, in which the Pb2+ ion not only interacts with the phosphate group but also with the carbonyl group of the nucleobase moiety, by folding of the mononucleotides. The structures thus characterized therefore differ from those proposed in solution and deduced from potentiometric studies. Our data also suggest that losing the nucleobase residue under CID conditions does not necessarily mean a lack of interaction between the metal and the nucleobase moiety, as commonly mentioned in the literature for large oligonucleotides.

Fragmentation behavior of Amadori‐peptides obtained by non‐enzymatic glycosylation of lysine residues with ADP‐ribose in tandem mass spectrometry

Mono- and poly-adenosine diphosphate (ADP)-ribosylation are common post-translational modifications incorporated by sequence-specific enzymes at, predominantly, arginine, asparagine, glutamic acid or aspartic acid residues, whereas non-enzymatic ADP-ribosylation (glycation) modifies lysine and cysteine residues. These glycated proteins and peptides (Amadori-compounds) are commonly found in organisms, but have so far not been investigated to any great degree. In this study, we have analyzed their fragmentation characteristics using different mass spectrometry (MS) techniques. In matrix-assisted laser desorption/ionization (MALDI)-MS, the ADP-ribosyl group was cleaved, almost completely, at the pyrophosphate bond by in-source decay. In contrast, this cleavage was very weak in electrospray ionization (ESI)-MS. The same fragmentation site also dominated the MALDI-PSD (post-source decay) and ESI-CID (collision-induced dissociation) mass spectra. The remaining phospho-ribosyl group (formed by the loss of adenosine monophosphate) was stable, providing a direct and reliable identification of the modification site via the b- and y-ion series. Cleavage of the ADP-ribose pyrophosphate bond under CID conditions gives access to both neutral loss (347.10 u) and precursor-ion scans (m/z 348.08), and thereby permits the identification of ADP-ribosylated peptides in complex mixtures with high sensitivity and specificity. With electron transfer dissociation (ETD), the ADP-ribosyl group was stable, providing ADP-ribosylated c- and z-ions, and thus allowing reliable sequence analyses.

The Influence of Cisplatin on the Gas-Phase Dissociation of Oligonucleotides Studied by Electrospray Ionization Tandem Mass Spectrometry

cis-Diamminedichloroplatinum(II) (cisplatin, DDP) is a cornerstone of anticancer therapy and has become one of the most widely used drugs for the treatment of various epithelial malignancies. The cytotoxicity of cisplatin is mainly based upon its affinity to adjacent guanines in nucleic acids, resulting in the formation of 1,2-intrastrand adducts. In this study the gas-phase dissociation of DNA- and RNA-cisplatin adducts is investigated by electrospray ionization (ESI) tandem mass spectrometry (MS/MS). The fundamental mechanistic aspects of fragmentation are elucidated to provide the basis for the tandem mass spectrometric determination of binding motifs and binding sites of this important anticancer drug. It is shown that the binding of cisplatin to vicinal guanines drastically alters the gas-phase fragmentation behavior of oligonucleotides. The 3'-C-O bond adjacent to the GG base pair is preferentially cleaved, leading to extensive formation of the corresponding w-ion. This observation was even made for oligoribonucleotides, which usually tend to form c- and y-ions under CID conditions. The absence of complementary ions of equal abundance indicates that oligonucleotide-cisplatin adducts are following more than one dissociation pathway in the gas-phase. Several mechanisms that explain the increased cleavage of the 3'-C-O bond and the lack of the complementary a-ion are proposed. Results of additional MS/MS experiments on methylphosphonate-oligodeoxynucleotides confirm the proposed mechanisms.

Energy-Resolved Structural Details Obtained from Gangliosides

Gangliosides, a family of glycosphingolipids (GSLs) that comprise sialic acid residue(s), are an important class of molecules that exist on the outer surface of the plasma membrane. To assess the functions of a particular series of gangliosides that play important roles in brain functions, their structures and localizations need to be investigated. We studied the structures of these gangliosides by collision-induced dissociation using quadrupole ion-trap mass spectrometry. The dissociation processes were investigated in detail based on energy-resolved mass spectrometry using sodiated molecules. The decision of utilization of the positive mode was based on the assumption that it was the generally applicable method for GSLs, including neutral ones. In this investigation, sialic acid residues were esterified to stabilize the linkages and to generate multiple fragment ions for successful structural investigations. A detailed analysis of a series of sodiated species of gangliosides based on energy-resolved mass spectrometry revealed that the GM1-equivelent fragments generated from the precursor ions under low energy CID conditions had the structural characteristics of their individual precursors. It was suggested that this information will be useful in determining the structures of their precursor gangliosides.

Selective Detection of Specific Protein–Ligand Complexes by Electrosonic Spray–Precursor Ion Scan Tandem Mass Spectrometry

A novel mass spectrometric method for the selective detection of specific protein-ligand complexes is presented. The new method is based on electrosonic spray ionization of samples containing protein and ligand molecules, and mass spectrometric detection using the precursor ion scanning function on a triple quadrupole instrument. Mass-selected intact protein-ligand complex ions are subjected to fragmentation by means of collision-induced dissociation in the collision cell of the instrument, while the second mass analyzer is set to the m/z of protonated ligand ions or their alkali metal adducts. The method allows for the detection of only those ions which yield ions characteristic of the ligand molecules upon fragmentation. Since the scan range of first analyzer is set well above the m/z of the ligand ion, and the CID conditions are established to permit fragmentation of only loosely bound, noncovalent complexes, the method is specific to the detection of protein-ligand complexes under described conditions. Behavior of biologically specific and nonspecific complexes was compared under various instrumental settings. Parameters were optimized to obtain maximal selectivity for specific complexes. Specific and nonspecific complexes were found to show markedly different fragmentation characteristics, which can be a basis for selective detection of complexes with biological relevance. Preparation of specific and nonspecific complexes containing identical building blocks was attempted. Complex ions with identical stoichiometry but different origin showed the expected difference in fragmentation characteristics, which gives direct evidence for the different mechanism of specific versus nonspecific complex ion formation.

Endometrial Carcinoma Biomarker Discovery and Verification Using Differentially Tagged Clinical Samples with Multidimensional Liquid Chromatography and Tandem Mass Spectrometry

The utility of differentially expressed proteins discovered and identified in an earlier study (DeSouza, L., Diehl, G., Rodrigues, M. J., Guo, J., Romaschin, A. D., Colgan, T. J., and Siu, K. W. M. (2005) Search for cancer markers from endometrial tissues using differentially labeled tags iTRAQ and cleavable ICAT with multidimensional liquid chromatography and tandem mass spectrometry. J. Proteome Res. 4, 377-386) to discriminate malignant and benign endometrial tissue samples was verified in a 40-sample iTRAQ (isobaric tags for relative and absolute quantitation) labeling study involving normal proliferative and secretory samples and Types I and II endometrial cancer samples. None of these proteins had the sensitivity and specificity to be used individually to discriminate between normal and cancer samples. However, a panel of pyruvate kinase, chaperonin 10, and alpha1-antitrypsin achieved the best results with a sensitivity, specificity, predictive value, and positive predictive value of 0.95 each in a logistic regression analysis. In addition, three new potential markers were discovered, whereas two other proteins showed promising trends but were not detected in sufficient numbers of samples to permit statistical validation. Differential expressions of some of these candidate biomarkers were independently verified using immunohistochemistry.