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In several neurodegenerative disorders, the neuronal proteins tau and α-synuclein adopt aggregation-prone conformations capable of replicating within and between cells. To better understand how these conformational changes drive neuropathology, we compared the interactomes of tau and α-synuclein in the presence or the absence of recombinant fibril seeds. Human embryonic stem cells with an inducible neurogenin-2 transgene were differentiated into glutamatergic neurons expressing (1) WT 0N4R tau, (2) mutant (P301L) 0N4R tau, (3) WT α-synuclein, or (4) mutant (A53T) α-synuclein, each genetically fused to a promiscuous biotin ligase (BioID2). Neurons expressing unfused BioID2 served as controls. After treatment with fibrils or PBS, interacting proteins were labeled with biotin in situ and quantified using mass spectrometry via tandem mass tag labeling. By comparing interactions in mutant versus WT neurons and in fibril- versus PBS-treated neurons, we observed changes in protein interactions that are likely relevant to disease progression. We identified 45 shared interactors, suggesting that tau and α-synuclein function within some of the same pathways. Potential loci of shared interactions include microtubules, Wnt signaling complexes, and RNA granules. Following fibril treatment, physiological interactions decreased, whereas other interactions, including those between tau and 14-3-3 η, increased. We confirmed that 14-3-3 proteins, which are known to colocalize with protein aggregates during neurodegeneration, can promote or inhibit tau aggregation in vitro depending on the specific combination of 14-3-3 isoform and tau sequence.

Expansion of the genetic code with unnatural amino acids (Uaas) has significantly increased the chemical space available to proteins for exploitation. Due to the inherent limitation of translational machinery and the required compatibility with biological settings, function groups introduced via Uaas to date are restricted to chemically inert, bioorthogonal, or latent bioreactive groups. To break this barrier, here we report a new strategy enabling the specific incorporation of biochemically reactive amino acids into proteins. A latent bioreactive amino acid is genetically encoded at a position proximal to the target natural amino acid; they react via proximity-enabled reactivity, selectively converting the latter into a reactive residue in situ. Using this Genetically Encoded Chemical COnversion (GECCO) strategy and harnessing the sulfur-fluoride exchange (SuFEx) reaction between fluorosulfate-l-tyrosine and serine or threonine, we site-specifically generated the reactive dehydroalanine and dehydrobutyrine into proteins. GECCO works both inter- and intramolecularly, and is compatible with various proteins. We further labeled the resultant dehydroalanine-containing protein with thiol-saccharide to generate glycoprotein mimetics. GECCO represents a new solution for selectively introducing biochemically reactive amino acids into proteins and is expected to open new avenues for exploiting chemistry in live systems for biological research and engineering.

Chemical cross-linking mass spectrometry (CXMS) is being increasingly used to study protein assemblies and complex protein interaction networks. Existing CXMS chemical cross-linkers target only Lys, Cys, Glu, and Asp residues, limiting the information measurable. Here we report a "plant-and-cast" cross-linking strategy that employs a heterobifunctional cross-linker that contains a highly reactive succinimide ester as well as a less reactive sulfonyl fluoride. The succinimide ester reacts rapidly with surface Lys residues "planting" the reagent at fixed locations on protein. The pendant aryl sulfonyl fluoride is then "cast" across a limited range of the protein surface, where it can react with multiple weakly nucleophilic amino acid sidechains in a proximity-enhanced sulfur-fluoride exchange (SuFEx) reaction. Using proteins of known structures, we demonstrated that the heterobifunctional agent formed cross-links between Lys residues and His, Ser, Thr, Tyr, and Lys sidechains. This geometric specificity contrasts with current bis-succinimide esters, which often generate nonspecific cross-links between lysines brought into proximity by rare thermal fluctuations. Thus, the current method can provide diverse and robust distance restraints to guide integrative modeling. This work provides a chemical cross-linker targeting unactivated Ser, Thr, His, and Tyr residues using sulfonyl fluorides. In addition, this methodology yielded a variety of cross-links when applied to the complex Escherichia coli cell lysate. Finally, in combination with genetically encoded chemical cross-linking, cross-linking using this reagent markedly increased the identification of weak and transient enzyme-substrate interactions in live cells. Proximity-dependent cross-linking will dramatically expand the scope and power of CXMS for defining the identities and structures of protein complexes.

Recombinant monoclonal antibodies are an important class of therapeutic agents that have found widespread use for the treatment of many human diseases. Here, we have examined the utility of ion mobility mass spectrometry (IMMS) for the rapid characterization of disulfide variants in intact IgG2 monoclonal antibodies. It is shown that IMMS reveals 2 to 3 gas-phase conformer populations for IgG2s. In contrast, a single gas-phase conformer is revealed using IMMS for both an IgG1 antibody and a Cys-232 [right arrow] Ser mutant IgG2, both of which are homogeneous with respect to disulfide bonding. This provides strong evidence that the observed IgG2 gas-phase conformers are related to disulfide bond heterogeneity. Additionally, IMMS analysis of redox enriched disulfide isoforms allows assignment of the mobility peaks to established disulfide bonding patterns. These data clearly illustrate how IMMS can be used to quickly provide information on the higher order structure of antibody therapeutics. 10.1021/ac1013139

The use of gas phase additives to stabilize noncovalent protein complexes in electrospray ionization mass spectrometry (ES-MS) is demonstrated for two protein--ligand interactions, an enzyme--small molecule inhibitor complex, and a protein--disaccharide complex. It is shown that the introduction of gas phase imidazole into the ES ion source effectively protects gas phase protein--ligand complexes against in-source dissociation. The stabilizing effect of imidazole vapor is comparable to that observed upon addition of imidazole to the ES solution. The introduction of sulfur hexafluoride, at high partial pressure, into the source region also effectively suppresses in-source dissociation of protein complexes. It is proposed that evaporative cooling is the primary mechanism responsible for the stabilizing effects observed for the gas phase additives.

Gas-phase ion/molecule chemistry has been combined with ion mobility separation and time-of-flight mass spectrometry to enable the characterization of large poly(ethylene glycol)s (PEGs) and PEGylated molecules (> 40 kDa). A facile method is presented in which gas-phase superbases are reacted in the high-pressure source region of commercial TOF mass spectrometers to manipulate the charge states of large ions generated by electrospray ionization (ESI). Charge stripping decreases the spectral congestion typically observed in ESI mass spectra of high molecular weight polydisperse PEGylated molecules. From these data, accurate average molecular weights and molecular weight distributions for synthetic polymers and PEGylated proteins are determined. The average MW measured for PEGylated Granulocyte colony-stimulating factor (rh-GCSF, 40 726.2 Da) is in good agreement with the theoretical value, and a 16 Da mass shift is easily observed in the spectrum of an oxidized form of the heterogeneous PEGylated protein. Ion mobility separations can fractionate PEGs of different chain length; when coupled with charge stripping ion/molecule reactions, ion mobility mass spectrometry (IMMS) offers several analytical advantages over mass spectrometry alone for the characterization of large PEGylated molecules including enhanced dynamic range, increased sensitivity, and specificity. Low abundance free PEG in a PEGylated peptide preparation, which is not directly detectable by mass spectrometry, can be easily observed and accurately quantified with gas-phase ion/molecule chemistry combined with ion mobility mass spectrometry.

Two new applications using multiple ion isolations in the cell of a Fourier transform-ion cyclotron resonance mass spectrometer equipped with an electrospray ionization source are described. A procedure that uses multiple ion isolations of an analyte and calibrants for internal calibration at each stage in a M[S.sup.n] experiment, under high-resolution exact-mass conditions, for structural characterization/elucidation of angiotensin I and rapamycin is illustrated. Fragment ion mass accuracies

Peptide ions are used as thermometer ions to quantitatively characterize the internal energy deposited by collisionally activated dissociation (CAD). The sustained off-resonance irradiation (SORI)-CAD dissociation kinetics of several peptide ions are also measured. The effective temperature of the ion population activated by SORI is obtained by a combination of Arrhenius activation parameters measured by blackbody infrared radiative dissociation.

Blackbody infrared radiative dissociation in a Fourier transform mass spectrometer was used to analyze the dissociation kinetics of a series of complementary and noncomplementary DNA duplexes. The dissociation of the duplexes resulted in cleavage of the noncovalent bonds and/or cleavage of covalent bonds leading to loss of a neutral nucleobase followed by backbone cleavage. Evidences were presented which indicate that Watson-Crick base pairing is preserved in complementary DNA duplexes in the gas phase.

A new method that makes possible, for the first time, simultaneous acquisition of individual dissociation mass spectra of isomeric ions in mixtures is presented. This method exploits the exquisite sensitivity of blackbody infrared radiative dissociation kinetics to minor differences in ion structure. Instead of separating precursor ions based on mass (isomers have identical mass), fragment ions are related to their original precursor ions on the basis of rate constants for dissociation. Mixtures of the peptide isomers des-[R.sup.1] and des-[R.sup.9] bradykinin are dissociated simultaneously at several temperatures. By fitting the kinetic data to double-exponential functions, the dissociation rate constant and abundance of each isomer in the mixture are obtained. To overcome the difficulty of fitting double-exponential functions, a novel global analysis method is used in which several dissociation data sets collected at different temperatures are simultaneously fit. The kinetic data measured at multiple temperatures are modeled with the preexponentials (corresponding to the abundance of each isomer) as 'global' parameters which are constant for all data sets and the exponentials (rate constants) as 'local' variables which differ for each data set. The use of global parameters significantly improves the accuracy with which abundances and dissociation rate constants of each individual compound can be obtained from the mixture data. Fragment ions produced from a mixture of these two isomers are related back to their respective precursor ions from the kinetic data. Thus, not only can the composition of the isomeric mixture be determined but an individual tandem mass spectrum of each component in the mixture can be obtained.

Blackbody infrared radiative dissociation spectra of the [(M + 5H).sup.5+] through [(M + 11H).sup.11+] ions of the protein ubiquitin (8.6 kDa) formed by electrospray ionization were measured in a Fourier-transform mass spectrometer. The 5+ ion dissociates exclusively by loss of water and/or ammonia, whereas the 11+ charge state dissociates only by formation of complementary y and b ions. These two processes are competitive for intermediate charge state ions, with the formation of y and b ions increasingly favored for the higher charge states. The y and b ions are formed by cleavage of the backbone amide bond on the C-terminal side of acidic residues exclusively, with cleavage adjacent to aspartic acid favored. Thermal unimolecular dissociation rate constants for the dissociation of each of these charge states were measured. From the temperature dependence of these rates, Arrhenius activation parameters in the rapid energy exchange limit are obtained. The activation energies ([E.sub.a]) and preexponential factors (A) for the 5+, 8+, and 9+ ions are 1.2 eV and [10.sup.12] [s.sup.-1], respectively. These values for the 6+ and 7+ ions are 0.9-1.0 eV and [10.sup.9] [s.sup.-1], and those for the 10+ and 11+ ions are 1.6 eV and [10.sup.16]-[10.sup.17] [s.sup.-1]. Thus, with the exception of the 5+ ion, the higher charge states of ubiquitin have larger dissociation activation energies than the lower charge states. The different A factors observed for production of y and b ions from different precursor charge states indicate that they are formed by different mechanisms, ranging from relatively complex rearrangements to direct bond cleavages. These results clearly demonstrate that the relative dissociation rates of large biomolecule ions by themselves are not necessarily a reliable indicator of their relative dissociation energies, even when similar fragment ions are formed.

Molecular activation by blackbody photons has a significant part in the unimolecular dissociation of large ions trapped at low pressure in a Fourier-transform mass spectrometer. Molecular ions of the protein ubiquitin equilibrate with the blackbody radiation field inside the vacuum chamber. The internal energy of a population of these ions is given by a Boltzmann distribution. From the temperature dependence of unimolecular dissociation rate constants measured in the zero-pressure limit, Arrhenius activation parameters equal to those in the high-pressure limit are derived.

A measurement of blackbody infrared radiative dissociation spectra of singly and doubly protonated bradykinin and its analogues revealed a dramatic difference in the activation parameters even for formation of similar fragment ions. These parameters were also found to be a highly sensitive probe of structure. They also reflect the effects of intramolecular electrostatic interactions in the doubly protonated ion. Results provide the first experimental evidence that a a salt-bridge structure is the most stable form of singly protonated bradykinin.

For large, multiply protonated gas-phase cytochrome c ions the Coulomb energy, dielectric polarizability and gas-phase basicities (GB) are calculated. The apparent GB (GBapp) depends on the protein conformation and for the 8+ charge state a difference of 43 kilocalorie/mol in GBapp of native and denatured ion is noticed. In a denatured 21+ ion, the Coulomb energy is 24 electro volts. This high electrostatic force of the highly protonated ions suggests their increased reactivity and dissociation.

The surface plasmon resonance spectroscopy technique is employed to determine the rate constants exhibited by the dissociation of different bovine [beta]-lactoglobulin compound with fatty acids. The results demonstrate that the kinetic stability of such interactions can be significantly reduced with the addition of water.

The study evidence for certain [gamma]-secretase modulators (GSMs) that target the 99-residue C-terminal domain (C99) of the amyloid precursor protein, a substrate of [gamma]-secretase, but not the protease complex itself is presented. The results from NMR demonstrated a lack of specific binding for GSMs to monodisperse C99 in LMPG micelles and nonspecific nature of bindings of GSMs to C99 aggregates.

The results of time-resolved thermal dissociation measurements and molecular dynamic simulations are reported for gaseous deprotonated ions of the specific complexes of bovine [beta]-lactoglobulin (Lg) and a series of the fatty acids (FA): C[H.sub.3][(C[H.sub.2]).sub.x]COOH. The obtained results suggested that the aliphatic chain of the FA remained bound within the hydrophobic cavity in the gas phase [(Lg + FA).sup.7] ions.

Chemical cross-linking mass spectrometry (CXMS) is being increasingly used to study protein assemblies and complex protein interaction networks. Existing CXMS chemical cross-linkers target only Lys, Cys, Glu, and Asp residues, limiting the information measurable. Here we report a "plant-and-cast" cross-linking strategy that employs a heterobifunctional cross-linker that contains a highly reactive succinimide ester as well as a less reactive sulfonyl fluoride. The succinimide ester reacts rapidlywith surface Lys residues "planting" the reagent at fixed locations on protein. The pendant aryl sulfonyl fluoride is then "cast" across a limited range of the protein surface, where it can react with multiple weakly nucleophilic amino acid sidechains in a proximityenhanced sulfur-fluoride exchange (SuFEx) reaction. Using proteins of known structures, we demonstrated that the heterobifunctional agent formed cross-links between Lys residues and His, Ser, Thr, Tyr, and Lys sidechains. This geometric specificity contrasts with current bis-succinimide esters, which often generate nonspecific cross-links between lysines brought into proximity by rare thermal fluctuations. Thus, the current method can provide diverse and robust distance restraints to guide integrative modeling. This work provides a chemical cross-linker targeting unactivated Ser, Thr, His, and Tyr residues using sulfonyl fluorides. In addition, this methodology yielded a variety of cross-links when applied to the complex Escherichia coli cell lysate. Finally, in combination with genetically encoded chemical crosslinking, cross-linking using this reagent markedly increased the identification of weak and transient enzyme-substrate interactions in live cells. Proximity-dependent cross-linking will dramatically expand the scope and power of CXMS for defining the identities and structures of protein complexes. [ABSTRACT FROM AUTHOR]