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Identification of proteins in complex mixtures by mass spectrometry is most useful when quantitative data is also obtained. We recently introduced isotope-coded affinity tags (ICAT reagents) for the relative quantification of proteins present in two or more biological samples. In this report, we describe a new generation of ICAT reagents that contain the following additional features:  (1) a visible tag that allows the electrophoretic position of tagged peptides during separation to be easily monitored; (2) a photocleavable linker that allows most of the tag to be removed prior to mass spectrometric analysis; (3) an isotope tag that contains carbon-13 and nitrogen-15 atoms instead of deuterium to ensure precise comigration of light and heavy tagged peptides by reverse-phase HPLC. These reagents contain an iodoacetyl group that selectively reacts with peptide cysteine residues. Peptide modification chemistry is also reported that allows tagging of peptides that are devoid of cysteine. The synthesis of these visible isotope-coded affinity tags (VICAT reagents), and their reaction with peptides are described in this report. VICAT reagents containing a carbon-14 visible probe or an NBD fluorophore are described. These reagents are most useful for the determination of the absolute quantity of specific target proteins in complex protein mixtures such as serum or cell lysates.

The 1-hydroxy-1-(N-methyl)aminoethyl radical (1) represents a simple model system for hydrogen atom adducts to the amide bond in gas-phase peptide and protein ions relevant to electron capture dissociation (ECD). Radical 1 was generated in the rarefied gas phase by femtosecond electron transfer to the stable cation prepared by selective O-protonation of N-methylacetamide. The main dissociations of 1 were loss of the hydroxyl hydrogen atom and the N-methyl group in a 1.7:1 ratio, as deduced from product analysis and deuterium labeling. The dissociations that occur on the 4.1 microsecond time scale are driven by large Franck−Condon effects on collisional electron transfer that deposit 93−103 kJ mol^-1 in the nascent radicals. Detailed analysis of the potential energy surface for dissociations of 1 revealed several conformers and isomeric transition states for dissociations of the O−H and N−CH3 bonds. The experimental branching ratio is in quantitative agreement with RRKM calculations within the accuracy of the G2 potential energy surface and favors cleavage of the O—H bond in 1 and loss of H. This finding contrasts previously reported results, as discussed in the paper.

Gas-phase protonation of carbonic acid is predicted to occur preferentially at the carbonyl oxygen with the 298 K proton affinity that was calculated as PA = 741 and 775 kJ mol^-1 for the syn- and anti-conformer, respectively. The hydroxyl groups are less basic with a topical PA = 660 kJ mol^-1. The standard enthalpy of formation of gas-phase carbonic acid was calculated as %@mt;sys@%Δ%@ital@%H%@rsf@%%@/xs;55;%lnwidth@%°%@/xs;63;(%lnwidth-x55)@%%@mh;-x63@%%@sb@%f,298%@sbx@%%@mx@% = −614 kJ mol^-1. Collisional neutralization of protonated carbonic acid, anti-1⁺, yields transient trihydroxymethyl radicals that dissociate rapidly by loss of a hydrogen atom, so that no survivor species are observed on a 360 ns time scale. High-level ab initio calculations with G2(MP2), G2, B3-MP2, and QCISD(T)/6-311+G(3df,2p) find two stable conformers of trihydroxymethyl radicals, anti-1 and syn-1, that interconvert rapidly by O−H bond rotations. The lowest-energy dissociation of anti-1 is loss of a hydrogen atom which requires 93 kJ mol^-1 in the transition state and forms the anti-conformer of carbonic acid with an overall reaction enthalpy, ΔHrxn,0 = 18 kJ mol^-1. Four other [C,H3,O3] isomers are found by calculations to be local energy minima, e.g., hydrogen-bonded complexes [O&dbd;C&sbd;O···H···OH2]^• (2) and [HO&sbd;H···O&dbd;C&sbd;OH]^• (3), and dihydroxymethoxy radicals anti-4 and syn-4. Of these, 2 was generated transiently in the gas phase and found to dissociate rapidly to water and CO2. Kinetic isotope effects on dissociations of anti-1 and syn-1 are analyzed by RRKM calculations and used to estimate the radical internal energy, which can be expressed as a simple sum of the cationic precursor internal energy and the energy gained by Franck−Condon effects on collisional electron transfer.

The bicoordinated dihydroxyphosphenium ion P(OH) 2+( 1+) was generated specifically by charge-exchange dissociative ionization of triethylphosphite and its connectivity was confirmed by collision induced dissociation and neutralization-reionization mass spectra. The major dissociation of 1+forming PO +ions at m/ z47 involved another isomer, OPOH 2+( 2+), for which the optimized geometry showed a long POH 2bond. Dissociative 70-eV electron ionization of diethyl phosphite produced mostly 1+together with a less stable isomer, HP(O)OH +( 3+). Ion 2+is possibly co-formed with 1+upon dissociative 70-eV electron ionization of methylphosphonic acid. Neutralization-reionization of 1+confirmed that P(OH) 2·( 1) was a stable species. Dissociations of neutral 1, as identified by variable-time measurements, involved rate-determining isomerization to 2followed by fast loss of water. A competitive loss of H occurs from long-lived excited states of 1produced by vertical electron transfer. The Aand Bstates undergo rate-determining internal conversion to vibrationally highly excited ground state that loses an H atom via two competing mechanisms. The first of these is the direct cleavage of one of the OH bonds in 1. The other is an isomerization to 3followed by cleavage of the PH bond to form OPOH as a stable product. The relative, dissociation, and transition state energies for the ions and neutrals were studied by ab initio and density functional theory calculations up to the QCISD(T)/6–311+G(3df,2p) and CCSD(T)/aug-cc-pVTZ levels of theory. RRKM calculations were performed to investigate unimolecular dissociation kinetics of 1. Excited state geometries and energies were investigated by a combination of configuration interaction singles and time-dependent density functional theory calculations.

The amino(hydroxy)methyl radical (1) represents the simplest model for hydrogen atom adducts to the amide bond. Radical 1 was generated in the gas phase by femtosecond electron transfer to protonated formamide and found to be stable on the microsecond time scale. The major unimolecular dissociation of 1 was loss of the hydrogen atom from the hydroxyl group. Losses of hydrogen atoms from the CH and NH2 groups in 1 were less abundant. RRKM calculations on the G2(MP2), G2, and CCSD(T)/aug-cc-pVTZ potential energy surfaces predicted preferential loss of the hydroxyl hydrogen atom, in qualitative agreement with experiment. Bimolecular reactions of hydrogen atoms with formamide were predicted by calculations to prefer H atom abstraction from the H−C bond forming H2 and NH2C&dbd;O^•. This reaction was calculated to be 43 kJ/mol exothermic and had to overcome an activation energy barrier of 28.5 kJ/mol. Hydrogen atom additions to the carbon and oxygen termini of the carbonyl group in formamide had similar activation energies, 51 and 49 kJ/mol, respectively. H atom addition to the C-terminus producing the aminomethyloxy radical (6) was calculated to be 2 kJ/mol endothermic.

The elusive N-hydroxypyridyl radical 1 has been generated transiently in the gas phase by collisional neutralization of the stable cation 1⁺. The radical underwent extensive dissociation by specific losses of H, OH, and ring-cleavage reactions, as elucidated by neutralization-reionization mass spectrometry aided by deuterium labeling. Effective QCISD(T)/6-311+G(3df,2p) and combined Møller−Plesset and density functional theory calculations indicated that loss of OH from 1 was 49 kJ mol^-1 exothermic and proceeded on the potential energy surface of the ground doublet electronic state of the radical. The loss of H and ring-cleavage dissociations were initiated by the formation of excited electronic states in 1 that provided the internal energy for these endothermic reactions. OH radical addition to pyridine was predicted by transition-state theory calculations to occur mainly (82%) in the C-3 and C-5 positions. Hydrogen atom addition to pyridine-N-oxide (2) was predicted to occur selectively at the oxygen atom and trigger a reaction sequence that can result in a highly exothermic catalytic isomerization of 2 to hydroxypyridines.

The uracil cation radical was calculated to exist predominantly as the 1,3-dioxo tautomer 1^{•+, similar to the most stable tautomer of neutral uracil (1). The enol forms of 1•+ were found to be 10−173 kJ mol-1 less stable than 1•+ and should not be significantly populated at 298 K thermal equilibrium. Cation radical 1•+ is a moderately strong gas-phase acid of topical acidities ΔHacid = 829, 921, 916, and 879 kJ mol-1 for the H-1, H-3, H-5, and H-6 protons, respectively. Ion 1•+ is capable of exothermic protonation of adenine, guanine, and cytosine, and of the arginine, lysine, histidine, and tryptophan amino acid residues in proteins. The hydrogen atom affinities of 1•+ were −ΔHrxn = 432, 371, and 360 kJ mol-1 for H-atom additions to O-4, O-2, and C-5, respectively. 1•+ was calculated to exothermically abstract the thiol hydrogen atom from CH3SH, the hydroxyl hydrogen from phenol, and an α-hydrogen atom from glycine N-methylamide. Uracil radicals formed by deprotonation of 1•+ were calculated to have large hydrogen atom affinities that should allow for exothermic abstraction of H-atoms from thiol groups, phenolic hydroxyls, and amino acid backbone α-methylene and methine groups. Protonation by a uracil cation radical followed by hydrogen atom abstraction can propagate radiation damage from the initial ionization site. In contrast to the highly reactive uracil cation radicals and radicals, the weakly bound uracil anion radical (1•-) was predicted to be much less reactive in the gas phase. Ion−molecule reactions of 1•- by proton and hydrogen atom abstractions from thiols, phenol, and α-positions of amino acids were calculated to be endothermic and thus very slow in the gas phase. 1•- can selectively deprotonate carboxylic groups as calculated for the reaction with glycine.}

The 4-hydroxy-3,4-dihydropyrimidine-2(1H)-on-4-yl radical (1), an elusive hydrogen atom adduct to the O-4 position in uracil, was generated in the gas phase by femtosecond collisional electron transfer to O-4 protonated uracil and investigated by neutralization−reionization mass spectrometry (NRMS). A fraction of radicals 1 was stable on the 5.1 μs time scale. The main unimolecular dissociations of 1 were ring cleavages and a specific loss of the hydrogen atom from O-4, as determined by deuterium labeling. Ab initio calculations up to effective QCISD(T)/6-311+G(3df,2p) and combined density functional theory and perturbational calculations up to B3-MP2/6-311+G(3df,2p) were used to obtain bond dissociation and transition state energies for several radical and ion dissociations. The energies were used for calculations of rate constants by RRKM and transition state theory. The dissociations observed by NRMS could not be explained by competitive reactions occurring on the potential energy surface of the ground doublet state of 1 and must to a large part originate from excited electronic states of 1 that are formed by vertical electron capture. The adiabatic ionization energy of 1 was calculated as 5.55 eV. Activation energies for hydrogen atom additions to uracil were found to inversely correlate with the addition exothermicities. The most reactive position in uracil was C-5 which was calculated to account for >95% hydrogen atom additions, followed by C-6 (<5% additions), while additions to O-2 and O-4 were kinetically negligible.

The elusive hydrogen atom adduct to the C-5 position in uracil was generated specifically in the gas phase, and its unimolecular dissociations were elucidated by neutralization−reionization mass spectrometry. Collisional electron transfer to the 5,6-dihydrouracil-6-yl cation generated the 5,6-dihydropyrimidine-2,4(1H,3H)-dion-6-yl radical (1), which was the most stable hydrogen atom adduct to C-5 in uracil. A substantial fraction of 1 was stable on the 5.1 μs time scale. The main unimolecular dissociations of 1 were specific losses of hydrogen atoms from C-5 and ring cleavages, as determined by deuterium labeling. Radical 1 did not isomerize unimolecularly to 5,6-dihydropyrimidine-2,4(1H,3H)-dion-5-yl (2). Ab initio calculations up to effective QCISD(T)/6-311+G(3df,2p) and combined density functional theory and perturbational calculations up to B3-MP2/6-311+G(3df,2p) provided bond dissociation and transition state energies for several radical and ion dissociations that were used for assessing rate constants by RRKM theory. The observed dissociations occur to a large part from excited electronic states of uracil radicals that are formed by vertical electron capture. The adiabatic ionization energies of uracil and 1 were calculated as 9.24 and 6.70 eV, respectively.

The dehydrobenzonitrilium distonic radical cations a−c, isomers of benzonitrile radical cation d, were prepared by collisional dehalogenation of protonated halogenobenzonitriles and characterized by tandem mass spectrometry (MS/MS/MS experiments and ion−molecule reactions) making use of a hybrid mass spectrometer of sector−quadrupole−sector configuration. These experimental results were further supported by density functional theory calculations at the B3LYP/6-311++G(3df,2p) level, indicating that although the distonic ions are less stable than ion d by ca. 45−50 kJ mol^-1, they are protected against isomerization by relatively large energy barriers. Relative energies of benzonitrile and its isomers as well as their proton affinities and ionization energies were also evaluated. N-protonation in benzonitrile is about 120 kJ mol^-1 more exothermic than ring protonation.

We describe the design and synthesis of substrate and internal standard conjugates for application in profiling enzyme activity of the enzymes α-d-2-deoxy-2-N-sulfonamido-glucosamine sulfamidase, α-d-2-deoxy-2-N-acetyl-glucosamine hydrolase, acetyl-coenzymeA:α-d-2-deoxy-2-amino-glucosamine transferase, and α-d-2-deoxy-2-N-acetyl-glucosamine-6-sulfate sulfatase. Deficiency of any one of these enzymes results in a single clinical phenotype known as Sanfilippo syndrome. Such substrates have been proven effective in the confirmation of enzyme deficiency by a combination of affinity chromatography (AC) and electrospray ionization mass spectrometry (ESIMS), which forms the foundation for a new analytical technology (ACESIMS) of general interest and application to clinical and biomedical research.

Nitromethyl radical (1) was generated in the gas phase by collisional electron detachment from the stable anion CH2NO2^- (1^-) and characterized by neutralization−reionization mass spectrometry. Radical 1 was stable on the 3.7 μs time scale. Laser electron photodetachment was also used to generate 1 and probe it by collisional reionization and mass spectrometry in a new experiment denoted as ^-PR⁺MS. The heat of formation of 1 was obtained by G2(MP2) and G2 calculations of enthalpies of atomization, isodesmic, and dissociation reactions as 126 kJ mol^-1, in excellent agreement with the value from CCSD(T)/aug-cc-pVTZ calculations, ΔH° f,298(1) = 127 kJ mol^-1. The dissociation energy of the C−H bond in nitromethane was calculated as 425 kJ mol^-1. No stable structure was found for singlet cation 1⁺ in accord with complete dissociation of vertically ionized 1⁺. Several anionic, radical, and cationic isomers of the general formula [C,H2,N,O2] were identified by ab initio calculations and investigated experimentally. CH2ONO^• (2) was generated transiently from the stable cation 2⁺ and found to dissociate completely to CH2O and NO on the 3.7 μs time scale. Radical 2 is bound by only 2 kJ mol^-1. Gas-phase deprotonation of methyl nitrite and N-hydroxyformamide, and fluoride anion-induced desilylation of (CH3)3SiCH2ONO produced anions with the O−CH&dbd;N−OH^- (4^-) and/or O&dbd;CH−NH−O^- (5^-) structures that were used to generate stable radicals 4 and/or 5. Collisional neutralization of stable O=C−NH−OH⁺ (6⁺) formed transiently radical 6 that dissociated exothermically to CO and HNOH^•. Cations 4⁺ and 6⁺ were found to partially interconvert following collisional ionization of radicals 4 and 6.

Several isomers of nitrobenzene (1) and nitrobenzene cation-radical (1^+•) were identified as stable structures and their gas-phase chemistry was investigated by the methods of tandem mass spectrometry and combined ab initio and density functional theory calculations. The relative energies of [C6,H5,N,O2] neutral molecules followed the order o-nitrosophenol (5, most stable) < p-nitrosophenol (7) < o-benzoquinonenitrone (10) < p-benzoquinoneoxime (19) < o-benzoquinoneoxime (11) < m-nitrosophenol (6) < nitrobenzene (1) < phenyl nitrite (8) < benzo[d]-1,3-dioxa-2-azolidine (13) ≪ triplet didehydrocyclohexa-2,4-dien-1-yliden-1-nitronic acids, para ((³A‘ ‘) 4) ≈ meta ((³A‘ ‘) anti-3) < ortho ((³A‘ ‘) syn-2) < singlet didehydrocyclohexa-2,4-dien-1-yliden-1-nitronic acids, meta ((¹A) syn-3) < ortho ((¹A) syn-2) < para ((¹A) 4) (least stable). Molecules 1, 5, 7, and 8, were generated in the gas phase by femtosecond collisional electron transfer and their dissociations were investigated by neutralization-reionization mass spectrometry. The relative energies of [C6,H5,N,O2]^+• cation radicals followed the order 7^+•(most stable) < 5^+• < 6^+• < anti-11^+• < 8^+• < 10^+• < 13^+• < benzo[d]-1,2-dioxa-3-azolidine (12^+•) < 1^+• < 4^+• < syn-3^+• < syn-2^+• (least stable).

Isomeric radicals corresponding to hydrogen atom adducts to 2-hydroxypyridine (1) and 2-(1H)pyridone (2) were investigated by neutralization-reionization mass spectrometry and combined ab initio and density functional theory calculations. Gas-phase protonation of 1 and 2 occurred preferentially at the nitrogen and oxygen atoms, respectively, to give a single 2-hydroxypyridinium ion 3⁺. The calculated topical proton affinities in 1 were 922, 602, 777, 649, 786, 694, and 746 kJ mol^-1 for the N-1, C-2, C-3, C-4, C-5, C-6, and OH positions, respectively. The topical proton affinities in 2 were 756, 824, 815, 692, and 930 kJ mol^-1 for the N-1, C-3, C-5, C-6, and carbonyl oxygen positions, respectively. The 2-hydroxy-(1H)pyridinium radical (3^•) was generated by collisional neutralization of ion 3⁺ and found to be stable on a 4.67 μs time scale. Radical 3^• dissociated by losses of the hydroxyl and amine hydrogen atoms and by ring cleavages. MP2 and B3LYP calculations with the 6-311G(2d,p) basis set established the 298 K relative energies of hydrogen atom adducts derived from 1 and 2 as 3-H-2 (11^•, most stable, 0) < 6-H-2 (14^•, +20) < 3H-1 (5^•,+37) < 4H-2 (12^•,+59) < 5H-2 (13^•,+60) < 5H-1 (7^•,+62) < 3^• (+67) < 6H-1 (8^•,+76) < 4H-1 (6^•,+86) < 2H-1 (4^•,+107) < 1H-2 (10^•,+139 kJ mol^-1). Hydrogen atom adducts to C-2 in 2 and to the hydroxyl group in 1 were found to be unstable and dissociated by ring opening and loss of H, respectively. RRKM calculations on the effective QCISD(T)/6-311+G(2d,p) potential energy surface showed cleavages of the O−H and N−H bonds in 3^• to be the lowest energy dissociations occurring in a 10:1 ratio. 1 was calculated to be 4.7 kJ mol^-1 more stable than 2 in the gas phase at 0 K. Fitting the experimental and calculated isotope effects on dissociations of deuterium-labeled radicals yielded a distribution function for the internal energy in the ground electronic state of 3^• formed by collisional electron transfer. The maximum of the internal energy distribution in ground-state 3^• (129 kJ mol^-1) was found to be expressed accurately by a combination of the internal energy of the precursor ion and the Franck−Condon energy gained on vertical electron transfer. The three lowest excited electronic states in 3^• were found by CIS/6-311G(2d,p) calculations to be outer states resulting from excitation of the unpaired electron in 3^• or electron capture by 3⁺. The energetics and radiative lifetimes of the outer excited states of 3^• allowed interpretation of the highly endothermic ring-cleavage dissociations. The unimolecular chemistry of 3 can be explained by a bimodal energy distribution due to the formation of the ground and excited electronic states upon femtosecond collisional electron transfer.

The title radicals were produced by femtosecond collisional electron transfer in the gas phase and studied by the methods of variable-time neutralization−reionization mass spectrometry combined with fast-beam laser photoexcitation and G2(MP2) ab initio/RRKM calculations. The methylsulfonyl radical (CH3SO2^•, 1) was calculated to be bound by 59 kJ mol^-1 against the lowest-energy dissociation to CH3^• and SO2 at 0 K and to have a heat of formation ΔHf,298(1) = −211 ± 4 kJ mol^-1. When formed by vertical electron transfer, radical 1 dissociated rapidly due to a large Franck−Condon energy, EFC = 141 kJ mol^-1. The reverse addition of CH3^• to the sulfur atom in SO2 had a potential energy barrier of 1.3 kJ mol^-1 and Arrhenius parameters, log A = 12.19 and Ea = 5.4 kJ mol^-1. The calculated addition rate constant, k295 = 1.7 × 10¹¹ cm³ mol^-1 s^-1, was in excellent agreement with the previous measurement of Simons et al. The methoxysulfinyl radical (CH3OSO^•, 2) was calculated to exist as an equilibrium mixture of syn (2s) and anti (2a) conformers. The Boltzmann-averaged heat of formation of 2 was calculated as ΔHf,298(2) = −230 ± 4 kJ mol^-1. Vertical neutralization of ions 2s⁺ and 2a⁺ produced substantial fractions of stable 2s,a. Dissociating 2s,a formed CH3^• and SO2 through unimolecular isomerization to 1. Direct dissociation of the C−O bond in 2s,a to form CH3^• and SO2 was calculated to have a large activation barrier (152 kJ mol^-1 from 2a) and did not compete with the isomerization to 1, which required 111 kJ mol^-1 from 2a. Photoexcitation of 2s,a resulted in a slightly increased formation of 2s,a⁺. This was interpreted with the help of CIS/6-311+G(3df,2p) calculations as being due to the formation of a bound excited B state of 2s upon electron transfer. The B state was photoexcited at 488 and 514.5 nm to high Rydberg states which were predicted to have large cross sections for collisional ionization. The A state of 2s was calculated to be bound but photoinactive. The C through E states of 2s were unbound and predicted to dissociate exothermically to CH3OS and (³P)O.

Variable-time neutralization−reionization mass spectrometry was used to generate elusive radicals CH3NO2H^• (1), CH3O(H)−NO^• (2) CH3O−N(H)−O^• (3), and CH3O−N−OH^• (4) that represent hydrogen atom adducts to nitromethane and methyl nitrite, respectively. Radicals 1, 3, and 4 were stable species on the 3.7 μs time scale of the experiment. Radical 2 dissociated completely to CH3OH and NO. The major unimolecular dissociations of 1 formed CH3NO + OH^• and CH3^• + NO2H, whereas loss of H^• to form CH3NO2 was insignificant. Gaussian 2(MP2) and density functional theory calculations provided the structures and bond dissociation energies of 1−4. The relevant parts of the potential-energy surface were used for RRKM and transition-state theory calculations of unimolecular dissociation kinetics. The calculated branching ratios for 1 were in accord with experiment. The unimolecular chemistry of radicals 1−4 formed by femtosecond collisional electron transfer can be accounted for by the properties of the ground electronic states of 1−4.

A series of isomeric 4-aminopyrimidinium radicals were used to model hydrogen atom adducts of nucleobases containing the 4-aminopyrimidine structure motif. Relative stabilities and activation energies for dissociations by hydrogen atom loss have been calculated by density functional theory and ab initio methods up to effective QCISD(T)/6-311+G(2d,p) for 4-amino-N-1-H− (1), 2-H− (2), N-3-H− (3), 4-H− (4), 5-H− (5), and 6-H− (6) pyrimidinium radicals and the 4-pyrimidylammonium radical (7). All these radicals were found to be bound species existing in potential energy wells. The order of stabilities has been established as 5 (most stable) > 3 > 2 > 1 > 6 > 4 ≫ 7 (least stable). Dissociations of the N−H and C−H bonds in 1-7 required activation barriers above the dissociation thresholds. RRKM calculations of unimolecular rate constants for N−H bond dissociations in 1 and 3 predicted substantial stabilization of these radicals by kinetic shift in the gas phase. Additions of hydrogen atoms to the N-1, C-2, N-3, C-4, C-5, and C-6 ring positions in 4-aminopyrimidine were found to be exothermic by 68, 70, 76, 23, 91, and 62 kJ mol^-1 at 0 K, respectively. Hydrogen atom addition to the NH2 group was 58 kJ mol^-1 endothermic. The activation barriers for the hydrogen atom additions to 4-aminopyrimidine were found to inversely correlate with the reaction enthalpies. The calculated rate constants predicted predominant (95%) hydrogen atom addition to C-5. The other positions were substantially less reactive, e.g., N-3 (2%), C-2 (1%), C-6 (0.8%), and N-1 (0.4%).

Hypervalent dimethylammonium radical, (CH3)2NH2^•, and its deuterium-labeled isotopomers (CH3)2ND2^•, (CH3)2NHD^•, (CD3)2NH2^•, and (CD3)2ND2^• were generated as transient species by collisional neutralization of their cations in the gas phase and studied by neutralization−reionization mass spectrometry, laser photoexcitation, and ab initio theory. (CH3)2ND2^•, (CH3)2NHD^•, and (CD3)2NH2^• gave fractions of metastable species of ≥3.3 μs lifetimes, whereas (CH3)2NH2^• and (CD3)2ND2^• dissociated completely on the same time scale. Metastable (CD3)2NH2^• and (CH3)2ND2^• were photoexcited but not photoionized with the combined 488 and 514.5 nm lines from an Ar-ion laser. Ab initio calculations with effective PMP4(SDTQ)/6-311++G(3df,2p) identified the (&Xtilde;)²A1 ground state of vertical ionization energy, IEv = 3.70 eV. RRKM calculations on the ab initio potential energy surface of the (&Xtilde;)²A1 state predicted predominant N−H and N−D bond dissociations but did not allow for competitive loss of CH3 or CD3. The four lowest excited states of (CH3)2NH2^•, (Ã)²B1, (&Btilde;)²A1, (&Ctilde;)²B2, and (&Dtilde;)²A1, were characterized by CIS/6-311++G(3df,2p) calculations, and their vertical ionization energies were calculated as 2.86, 2.57, 2.48, and 1.82 eV, respectively. The excited states were calculated to be strongly bound with respect to N−H bond dissociations. The N−C bond dissociations were interpreted by potential energy surface crossing of the &Btilde; and à states and transitions via conical intersection to the dissociative ground state.