Low Stoichiometry Research Articles

Phosphoproteomics technologies and applications in plant biology research.

Protein phosphorylation has long been recognized as an essential mechanism to regulate many important processes of plant life. However, studies on phosphorylation mediated signaling events in plants are challenged with low stoichiometry and dynamic nature of phosphorylated proteins. Significant advances in mass spectrometry based phosphoproteomics have taken place in recent decade, including phosphoprotein/phosphopeptide enrichment, detection and quantification, and phosphorylation site localization. This review describes a variety of separation and enrichment methods for phosphoproteins and phosphopeptides, the applications of technological innovations in plant phosphoproteomics, and highlights significant achievement of phosphoproteomics in the areas of plant signal transduction, growth and development.

Improving Blood Plasma Glycoproteome Coverage by Coupling Ultracentrifugation Fractionation to Electrostatic Repulsion-Hydrophilic Interaction Chromatography Enrichment.

Blood plasma is considered to be an excellent source of disease biomarkers because it contains proteins, lipids, metabolites, cell, and cell-derived extracellular vesicles from different cellular origins including diseased tissues. Most secretory and membranous proteins that can be found in plasma are glycoproteins; therefore, the plasma glycoproteome is one of the major subproteomes that is highly enriched with disease biomarkers. As a result, the glycoproteome has attracted much attention in clinical proteomic research. The modification of proteins with glycans regulates a wide range of functions in biology, but profiling plasma glycoproteins on a global scale has been hampered by the presence of low stoichiometry of glycoproteins in a complex high abundance plasma proteome background and lack of effective analytical technique. This study aims to improve plasma glycoproteome coverage using pig plasma as a model sample with a two-step strategy. The first step involves fractionation of the plasma proteins using ultracentrifugation into supernatant and pellet that is believed to contain low abundant glycoproteins. In the second step, further enrichment of glycopeptides was achieved in both fractions by adopting electrostatic repulsion hydrophilic interaction chromatography (ERLIC) coupled to tandem mass spectrometry (LC-MS/MS) analysis. The coverage of enriched glycoproteins in supernatant, pellet, and whole plasma sample as control was compared. Using this simple sample fractionation approach by ultracentrifugation and further ERLIC enrichment technique, sample complexity was reduced and glycoproteome coverage was significantly enhanced in supernatant and pellet fractions (by >50%) compared with whole plasma sample. This study showed that when ultracentrifugation is coupled to ERLIC glycopeptides enrichment and glycoproteome identification are significantly improved. This study demonstrates the combination of ultracentrifugation and ERLIC as a useful method for discovering plasma glycoprotein disease biomarkers.

Application of quantitative proteomics to the integrated analysis of the ubiquitylated and global proteomes of xenograft tumor tissues.

BackgroundPost-translational modification by ubiquitin is a fundamental regulatory mechanism that is implicated in many cellular processes including the cell cycle, apoptosis, cell adhesion, angiogenesis, and tumor growth. The low stoichiometry of ubiquitylation presents an analytical challenge for the detection of endogenously modified proteins in the absence of enrichment strategies. The recent availability of antibodies recognizing peptides with Lys residues containing a di-Gly ubiquitin remnant (K-ε-GG) has greatly improved the ability to enrich and identify ubiquitylation sites from complex protein lysates via mass spectrometry. To date, there have not been any published studies that quantitatively assess the changes in endogenous ubiquitin-modification protein stoichiometry status at the proteome level from different tissues.ResultsIn this study, we applied an integrated quantitative mass spectrometry based approach using isobaric tags for relative and absolute quantitation (iTRAQ) to interrogate the ubiquitin-modified proteome and the cognate global proteome levels from luminal and basal breast cancer patient-derived xenograft tissues. Among the proteins with quantitative global and ubiquitylation data, 91 % had unchanged levels of total protein relative abundance, and less than 5 % of these proteins had up- or down-regulated ubiquitylation levels. Of particular note, greater than half of the proteins with observed changes in their total protein level also had up- or down-regulated changes in their ubiquitylation level.ConclusionsThis is the first report of the application of iTRAQ-based quantification to the integrated analysis of the ubiquitylated and global proteomes at the tissue level. Our results underscore the importance of conducting integrated analyses of the global and ubiquitylated proteomes toward elucidating the specific functional significance of ubiquitylation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12014-015-9086-5) contains supplementary material, which is available to authorized users.

Peptide-polymer ligands for a tandem WW-domain, an adaptive multivalent protein-protein interaction: lessons on the thermodynamic fitness of flexible ligands.

Three polymers, poly(N-(2-hydroxypropyl)methacrylamide) (pHPMA), hyperbranched polyglycerol (hPG), and dextran were investigated as carriers for multivalent ligands targeting the adaptive tandem WW-domain of formin-binding protein (FBP21). Polymer carriers were conjugated with 3–9 copies of the proline-rich decapeptide GPPPRGPPPR-NH2 (P1). Binding of the obtained peptide–polymer conjugates to the tandem WW-domain was investigated employing isothermal titration calorimetry (ITC) to determine the binding affinity, the enthalpic and entropic contributions to free binding energy, and the stoichiometry of binding for all peptide–polymer conjugates. Binding affinities of all multivalent ligands were in the µM range, strongly amplified compared to the monovalent ligand P1 with a KD > 1 mM. In addition, concise differences were observed, pHPMA and hPG carriers showed moderate affinity and bound 2.3–2.8 peptides per protein binding site resulting in the formation of aggregates. Dextran-based conjugates displayed affinities down to 1.2 µM, forming complexes with low stoichiometry, and no precipitation. Experimental results were compared with parameters obtained from molecular dynamics simulations in order to understand the observed differences between the three carrier materials. In summary, the more rigid and condensed peptide–polymer conjugates based on the dextran scaffold seem to be superior to induce multivalent binding and to increase affinity, while the more flexible and dendritic polymers, pHPMA and hPG are suitable to induce crosslinking upon binding.

Phospho-iTRAQ data article: Assessing isobaric labels for the large-scale study of phosphopeptide stoichiometry.

The ability to distinguish between phosphopeptides of high and low stoichiometry is essential to discover the true extent of protein phosphorylation. We here extend the strategy whereby a peptide sample is briefly split in two identical parts and differentially labeled preceding the phosphatase treatment of one part (Pflieger et al., 2008. Mol. Cell. Proteomics, 7: 326–46 [1]; Wu et al., 2011. Nat. Methods, 8: 677–83 [2]). Our Phospho-iTRAQ method focuses on the unmodified counterparts of phosphorylated peptides, which thus circumvents the ionization, fragmentation, and phospho-enrichment difficulties that hamper quantitation of stoichiometry in most common phosphoproteomics methods. Since iTRAQ enables multiplexing, simultaneous (phospho)proteome comparison between internal replicates and multiple samples is possible. The technique was validated on multiple instrument platforms by adding internal standards of high stoichiometry to a complex lysate of control and EGF-stimulated HeLa cells. To demonstrate the flexibility of PhosphoiTRAQ with regards to the experimental setup and data mining, the proteome coverage was extended through gel fractionation, while an internal replicate measurement creates more stringent data analysis opportunities. The latter allows other researchers to set their own threshold for selecting potential phosphorylation events in the dataset presented here, depending on the biological question or corroboration under investigation. The latest developments in MS instrumentation promise to further increase the resolution of the stoichiometric measurement of Phospho-iTRAQ in the future. The data accompanying the manuscript on this approach (Glibert et al., 2015, J. Proteome Res.14: 2015, 839–49 [5]) have been deposited to the ProteomeXchange with identifier PXD001574.

The anionic biosurfactant rhamnolipid does not denature industrial enzymes.

Biosurfactants (BS) are surface-active molecules produced by microorganisms. Their combination of useful properties and sustainable production make them promising industrial alternatives to petrochemical and oleochemical surfactants. Here we compare the impact of the anionic BS rhamnolipid (RL) and the conventional/synthetic anionic surfactant sodium dodecyl sulfate (SDS) on the structure and stability of three different commercially used enzymes, namely the cellulase Carezyme® (CZ), the phospholipase Lecitase Ultra® (LT) and the α-amylase Stainzyme® (SZ). Our data reveal a fundamental difference in their mode of interaction. SDS shows great diversity of interaction toward the different enzymes. It efficiently unfolds both LT and CZ, but LT is unfolded by SDS through formation of SDS clusters on the enzyme well below the cmc, while CZ is only unfolded by bulk micelles and on average binds significantly less SDS than LT. SDS binds with even lower stoichiometry to SZ and leads to an increase in thermal stability. In contrast, RL does not affect the tertiary or secondary structure of any enzyme at room temperature, has little impact on thermal stability and only binds detectably (but at low stoichiometries) to SZ. Furthermore, all enzymes maintain activity at both monomeric and micellar concentrations of RL. We conclude that RL, despite its anionic charge, is a surfactant that does not compromise the structural integrity of industrially relevant enzymes. This makes RL a promising alternative to current synthetic anionic surfactants in a wide range of commercial applications.

Dynamics of anode–cathode interaction in a polymer electrolyte fuel cell revealed by simultaneous current and potential distribution measurements under local reactant-starvation conditions

Low stoichiometry operation of fuel cells induces nonuniformities in the current generation and electrode potentials across the active electrode area. In this study, current and potential distributions are measured simultaneously in a fuel cell operating under reactant (fuel/air)-starvation conditions. During the galvanostatic operation under local reactant starvation, the localized increase of current density is observed closer to the inlet. Here under air starvation, cathode potentials dropped uniformly across the electrode area. However, during fuel starvation, a nonuniform cathode potential profile is observed. During the potentiostatic mode of operation under air-starvation condition, cathode potentials remained uniform and constant across the electrode area. However, under fuel starvation, the cathode potential profile is influenced by the anode potential profile. Electrode–electrode interaction especially during fuel starvation is captured by simultaneous measurements of current and potential distribution.

Development of a tandem affinity phosphoproteomic method with motif selectivity and its application in analysis of signal transduction networks.

Phosphorylation is an important post-translational modification that is involved in regulating many signaling pathways. Of particular interest are the growth factor mediated Ras and phosphoinositide 3-kinase (PI3K) signaling pathways which, if misregulated, can contribute to the progression of cancer. Phosphoproteomic methods have been developed to study regulation of signaling pathways; however, due to the low stoichiometry of phosphorylation, understanding these pathways is still a challenge. In this study, we have developed a multi-dimensional method incorporating electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) with tandem IMAC/TiO2 enrichment for subsequent phosphopeptide identification by LC/MS/MS. We applied this method to PDGF-stimulated NIH 3T3 cells to provide over 11,000 unique phosphopeptide identifications. Upon motif analysis, IMAC was found to enrich for basophilic kinase substrates while the subsequent TiO2 step enriched for acidophilic kinase substrates, suggesting that both enrichment methods are necessary to capture the full complement of kinase substrates. Biological functions that were over-represented at each PDGF stimulation time point, together with the phosphorylation dynamics of several phosphopeptides containing known kinase phosphorylation sites, illustrate the feasibility of this approach in quantitative phosphoproteomic studies.

Specific enrichment of phosphoproteins using functionalized multivalent nanoparticles.

Analysis of protein phosphorylation remains a significant challenge due to the low abundance of phosphoproteins and the low stoichiometry of phosphorylation, which requires effective enrichment of phosphoproteins. Here we have developed superparamagnetic nanoparticles (NPs) whose surface is functionalized by multivalent ligand molecules that specifically bind to the phosphate groups on any phosphoproteins. These NPs enrich phosphoproteins from complex cell and tissue lysates with high specificity as confirmed by SDS-PAGE analysis with a phosphoprotein-specific stain and mass spectrometry analysis of the enriched phosphoproteins. This method enables universal and effective capture, enrichment, and detection of intact phosphoproteins toward a comprehensive analysis of the phosphoproteome.

Facile Synthesis of Zirconia‐Modified Magnetic Nanoparticles for Purification of Phosphopeptides

AbstractBecause of the low stoichiometry from enzymatic hydrolysis of casein, purification of casein phosphopeptides (CPPs) is necessary prior to application in infant formula or nutritional foods. In this study, functional zirconia‐modified magnetic nanoparticles (Fe3O4@ZrO2) were prepared and applied for purification of CPPs. The physical shape, crystalline structures and chemical groups were characterized by transmission electron microscopy, X‐ray diffraction and Fourier transform infrared. Fe3O4@ZrO2 nanoparticles had nano‐dimension and good dispersion. The purification studies showed that the high selectivity of Fe3O4@ZrO2 was obtained at pH 4.5. Then the structures of reused Fe3O4@ZrO2 were characterized to study the stability of material. The purity of CPPs isolated by Fe3O4@ZrO2 was analyzed to study the affinity force of material. All results confirmed that Fe3O4@ZrO2 nanoparticles had good stability and high affinity force for CPPs after 15 repeated cycles, and the maximum loading amount of material was 105.53 mg/g with lower N/P (5.94) of CPPs.Practical ApplicationsCasein phosphopeptides can enhance the absorption and utilization of calcium, but it needs to be purified before used as calcium nutritional supplements. Most of existing purification methods are two‐step methods, which have limitations in food application. Compared with traditional methods, the separation method using Fe3O4@ZrO2 nanoparticles can one‐step purify phosphopeptides from real samples, and shows good performance. The purification process using Fe3O4@ZrO2 nanoparticles has the simpler procedures, higher enrichment efficiency and shorter production cycle. It means that the purification process is easy to expand the scale of production for improving the yield and quality of phosphopeptides, which can satisfy the demand of market. The results demonstrate that Fe3O4@ZrO2, which has good stability and high affinity force for metal oxide affinity peptides, is one new type of extraction materials and possesses wide application prospects in food industry.

Pressure distribution method for ex-situ evaluation of flow distribution in polymer electrolyte membrane fuel cells

Fuel cells for automotive applications consist of cells with large active areas. The active area is generally between 150 cm2 and 400 cm2. The reaction gases and the cooling media are distributed via bipolar plates to the reaction zones. Understanding local and cell wide gas distribution within the flow field at high current densities greater than 2.0 A/cm2 is a key factor regarding efficiency at low stoichiometry, lambda less than 2. In this paper a new method is introduced, which can be used as ex-situ evaluation of flow distribution. The gas pressure distribution is mapped with an array of 5 × 12 membrane differential pressure sensors by measuring the static pressure locally against the outlet pressure. Below a differential pressure of 100 mbar the signal measurement accuracy is ±2.5 mbar. This is demonstrated in a flow field with an active area of 250 cm2. The sensors are located next to the micro porous layer of the gas diffusion layer to avoid any impact of dynamic pressure. The effect of the intrusion of gas diffusion layer material into the flow channels on the fluid distribution is evaluated at clamping pressures between 0.6 MPa and 4.2 MPa.

Model development and optimization of operating conditions to maximize PEMFC performance by response surface methodology

Optimization of operating conditions to obtain maximum power in PEMFCs could have a significant role to reduce the costs of this emerging technology. In the present experimental study, a single serpentine PEMFC is used to investigate the effects of operating conditions on the electrical power production of the cell. Four significant parameters including cathode stoichiometry, anode stoichiometry, gases inlet temperature, and cathode relative humidity are studied using Design of Experiment (DOE) to obtain an optimal power. Central composite second order Response Surface Methodology (RSM) is used to model the relationship between goal function (power) and considered input parameters (operating conditions). Using this statistical–mathematical method leads to obtain a second-order equation for the cell power. This model considers interactions and quadratic effects of different operating conditions and predicts the maximum or minimum power production over the entire working range of the parameters. In this range, high stoichiometry of cathode and low stoichiometry of anode results in the minimum cell power and contrary the medium range of fuel and oxidant stoichiometry leads to the maximum power. Results show that there is an optimum value for the anode stoichiometry, cathode stoichiometry and relative humidity to reach the best performance. The predictions of the model are evaluated by experimental tests and they are in a good agreement for different ranges of the parameters.

Phospho-iTRAQ: assessing isobaric labels for the large-scale study of phosphopeptide stoichiometry.

The ability to distinguish between phosphopeptides of high and low stoichiometry is essential to discover the true extent of protein phosphorylation. We here extend the strategy whereby a peptide sample is briefly split in two identical parts and differentially labeled preceding the phosphatase treatment of one part. Our use of isobaric tags for relative and absolute quantitation (iTRAQ) marks the first time that isobaric tags have been applied for the large-scale analysis of phosphopeptides. Our Phospho-iTRAQ method focuses on the unmodified counterparts of phosphorylated peptides, which thus circumvents the ionization, fragmentation, and phospho-enrichment difficulties that hamper quantitation of stoichiometry in most common phosphoproteomics methods. Since iTRAQ enables multiplexing, simultaneous (phospho)proteome comparison between internal replicates and multiple samples is possible. The technique was validated on multiple instrument platforms by adding internal standards of high stoichiometry to a complex lysate of control and EGF-stimulated HeLa cells. To demonstrate the flexibility of Phospho-iTRAQ with regards to the experimental setup, the proteome coverage was extended through gel fractionation, while an internal replicate measurement created more stringent data analysis opportunities. The latest developments in MS instrumentation promise to further increase the resolution of the stoichiometric measurement of Phospho-iTRAQ in the future. The data have been deposited to the ProteomeXchange with identifier PXD001574.

Internal Humidification in PEM Fuel Cells Using Wick Based Water Transport

The feasibility of a wick-based technique for direct humidification of the membranes in proton exchange membrane fuel cells (PEMFC) under dry feed operation is investigated. The fuel cell is operated in several configurations using a wicking material over the active area facilitating water transport directly to the membrane electrode assembly (MEA) through capillary action. Among different configurations (A – D) tested, wick used for configuration-B in co-flow mode and configuration-C in counter-flow mode showed improved performance with dry gas feed in comparison to control configuration-A which was operated using conventional bubble humidification. It is further shown that at low air stoichiometry of 1.5 with wick, enhanced peak power density of 475 mW/cm2 was obtained without any external water supply to the wick in configuration-C under co-flow mode. The performance obtained due to the incorporation of wick in the conventional fuel cell system has been analyzed by means of electrochemical impedance measurements and estimating the Ohmic and charge transfer resistances in different configurations. It is shown that among different configurations, the Ohmic and charge transfer resistances corroborate the variation in fuel cell performance obtained from polarization curves.

Analysis of nitrated proteins in Saccharomyces cerevisiae involved in mating signal transduction.

Protein tyrosine nitration (PTN) is a PTM that regulates signal transduction and inflammatory responses, and is related to neurodegenerative and cardiovascular diseases. The cellular function of PTN remains unclear because the low stoichiometry of PTN limits the identification and quantification of nitrated peptides. Effective enrichment is an important aspect of PTN analysis. In this study, we analyzed the in vivo nitroproteome elicited by mating signal transduction in Saccharomyces cerevisiae using a novel chemical enrichment method followed by LC-MS/MS. Nitroproteome profiling successfully identified changes in the nitration states of 14 proteins during mating signal transduction in S. cerevisiae, making this the first reported in vivo nitroproteome in yeast. We investigated the biological functions of these nitroproteins and their relationships to mating signal transduction in S. cerevisiae using a protein-protein interaction network. Our results suggest that PTN and denitration may be involved in nonreactive nitrogen species-mediated signal transduction and can provide clues for understanding the functional roles of PTN in vivo.

A novel role for WAVE1 in controlling actin network growth rate and architecture.

Branched actin filament networks in cells are assembled through the combined activities of Arp2/3 complex and different WASP/WAVE proteins. Here we used TIRF and electron microscopy to directly compare for the first time the assembly kinetics and architectures of actin filament networks produced by Arp2/3 complex and dimerized VCA regions of WAVE1, WAVE2, or N-WASP. WAVE1 produced strikingly different networks from WAVE2 or N-WASP, which comprised unexpectedly short filaments. Further analysis showed that the WAVE1-specific activity stemmed from an inhibitory effect on filament elongation both in the presence and absence of Arp2/3 complex, which was observed even at low stoichiometries of WAVE1 to actin monomers, precluding an effect from monomer sequestration. Using a series of VCA chimeras, we mapped the elongation inhibitory effects of WAVE1 to its WH2 ("V") domain. Further, mutating a single conserved lysine residue potently disrupted WAVE1's inhibitory effects. Taken together, our results show that WAVE1 has unique activities independent of Arp2/3 complex that can govern both the growth rates and architectures of actin filament networks. Such activities may underlie previously observed differences between the cellular functions of WAVE1 and WAVE2.

Correlation of transducin photoaffinity labeling with the specific formation of intermolecular disulfide linkages in its α-subunit.

Transducin (T) is a heterotrimer of Tα, Tβ, and Tγ subunits. In the presence of light-activated rhodopsin, 8-azidoguanosine triphosphate (8-N3GTP) was covalently incorporated into T in a UV-light photodependent manner, with a low stoichiometry of 0.02 mol of 8-N3GTP per mol of T. Although Tα was preferentially labeled by 8-N3GTP, Tβ and Tγ were also modified. Photolabeling of T was specifically inhibited by GDP and GTP, but not by β,γ-imido-guanosine 5'-triphosphate (GMP-PNP), indicating that 8-N3GTP was modifying the GDP binding site of the holoenzyme. This was consistent with the observation that the photoaffinity probe was completely hydrolyzed to 8-N3GDP by T activated by illuminated rhodopsin. The formation of intermolecular disulfide associations in T was also determined because photolabeling of T was performed under non-reducing conditions. We established that Cys-347 of Tα was the major residue involved in the formation of disulfide-linked T oligomers. Other cysteines of Tα, such as Cys-321, also participated in the formation of disulfide bonds, revealing a complex pattern of intermolecular disulfide cross-links that led to the polymerization of T. The spontaneous generation of these cystines in Tα inhibited the light-dependent GTPase and GMP-PNP binding activities of T. A model was constructed illustrating that when two heterotrimers dimerize through the formation of disulfide bridges between the Cys-347 of their Tα subunits, the guanine ring of the 8-N3GDP bound to one T molecule might approach to the Tβγ-complex of the other heterotrimer. This model provides an explanation for the additional photolabeling of Tβ and Tγ by 8-N3GTP.

Proteomic approaches for site-specific O-GlcNAcylation analysis.

O-GlcNAcylation is a dynamic protein post-translational modification of serine or threonine residues by an O-linked monosaccharide N-acetylglucosamine (O-GlcNAc). O-GlcNAcylation was discovered three decades ago and its significance has been implicated in several disease states, such as metabolic diseases, cancer and neurological diseases. Yet it remains technically challenging to characterize comprehensively and quantitatively because of its low abundance, low stoichiometry and extremely labile nature under conventional collision-induced dissociation tandem MS conditions. Herein, we review the recent advances addressing these challenges in developing proteomic approaches for site-specific O-GlcNAcylation analysis, including specific enrichment of O-GlcNAc peptides/proteins, unambiguous site-determination of O-GlcNAc modification and quantitative analysis of O-GlcNAcylation.

High performance of nanoscaled Fe2O3 catalyzing UV-Fenton under neutral condition with a low stoichiometry of H2O2: Kinetic study and mechanism

Kinetics and mechanism of nano-Fe2O3 catalyzing UV-Fenton were investigated in this study with catechol as model pollutant. This type of heterogeneous UV-Fenton was proven to be characteristic of a wide pH range from mildly acidic to slightly alkaline and low H2O2 requirement accompanied with H2O2 utilization efficiency of larger than 1.0. Experiments of degradation under neutral condition with and without pH process control and analysis of several carboxylic acids were performed to find out the reason for treatment effectiveness under neutral condition. Kinetics study using the pseudo-first-order equation was performed to evaluate the effects of initial pH, H2O2 dose, nano-Fe2O3 dose, and reaction temperature on the rate constant of COD reduction, it was interesting to find the rate constant was similar among initial pH values from 3.0 to 8.0. Based on the data of rate constant under different temperatures, Ea (activation energy), ΔH (enthalpy change) and ΔS (entropy change) were calculated to be 45.1(±6.3) kJ/mol, 42.6(±6.3) kJ/mol and −148.0(±20.8) J/K/mol, respectively. Radical identification experiments based on inhibition of methylene blue degradation under respective scavenger for HO (hydroxyl radical), O21 (singlet oxygen) and O2− (superoxide radical) showed HO, O21 and O2− participated in nano-Fe2O3 catalyzing UV-Fenton. Presence of oxygen could greatly enhance the degradation rate compared with the case of bubbling nitrogen, and a possible mechanism of catechol oxidation in nano-Fe2O3 catalyzing UV-Fenton was proposed. At last, reusability and stability of nano-Fe2O3 were evaluated in six repeated runs. This study will provide some deep understanding of nano-Fe2O3 catalyzing UV-Fenton with respect to technical advantage and oxidation mechanism.

Phosphorylation Dependence and Stoichiometry of the Complex Formed by Tyrosine Hydroxylase and 14-3-3γ

Phosphorylated tyrosine hydroxylase (TH) can form complexes with 14-3-3 proteins, resulting in enzyme activation and stabilization. Although TH was among the first binding partners identified for these ubiquitous regulatory proteins, the binding stoichiometry and the activation mechanism remain unknown. To address this, we performed native mass spectrometry analyses of human TH (nonphosphorylated or phosphorylated on Ser19 (TH-pS19), Ser40 (TH-pS40), or Ser19 and Ser40 (TH-pS19pS40)) alone and together with 14-3-3γ. Tetrameric TH-pS19 (224 kDa) bound 14-3-3γ (58.3 kDa) with high affinity (Kd = 3.2 nM), generating complexes containing either one (282.4 kDa) or two (340.8 kDa) dimers of 14-3-3. Electron microscopy also revealed one major population of an asymmetric complex, consistent with one TH tetramer and one 14-3-3 dimer, and a minor population of a symmetric complex of one TH tetramer with two 14-3-3 dimers. Lower phosphorylation stoichiometries (0.15–0.54 phosphate/monomer) produced moderate changes in binding kinetics, but native MS detected much less of the symmetric TH:14-3-3γ complex. Interestingly, dephosphorylation of [32P]-TH-pS19 was mono-exponential for low phosphorylation stoichiometries (0.18–0.52), and addition of phosphatase accelerated the dissociation of the TH-pS19:14-3-3γ complex 3- to 4-fold. All together this is consistent with a model in which the pS19 residues in the TH tetramer contribute differently in the association to 14-3-3γ. Complex formation between TH-pS40 and 14-3-3γ was not detected via native MS, and surface plasmon resonance showed that the interaction was very weak. Furthermore, TH-pS19pS40 behaved similarly to TH-pS19 in terms of binding stoichiometry and affinity (Kd = 2.1 nM). However, we found that 14-3-3γ inhibited the phosphorylation rate of TH-pS19 by PKA (3.5-fold) on Ser40. We therefore conclude that Ser40 does not significantly contribute to the binding of 14-3-3γ, and rather has reduced accessibility in the TH:14-3-3γ complex. This adds to our understanding of the fine-tuned physiological regulation of TH, including hierarchical phosphorylation at multiple sites.