Activation of ZAP-70 through specific dephosphorylation at the inhibitory Tyr-292 by the low molecular weight phosphotyrosine phosphatase (LMPTP)

The ZAP-70 protein-tyrosine kinase plays a central role in signaling from the T cell antigen receptor. Recruitment and activation of ZAP-70 are transient and are terminated by phosphorylation of negative regulatory tyrosine residues and dephosphorylation of positively acting sites. We report that the low molecular weight protein-tyrosine phosphatase (LMPTP) specifically dephosphorylates the negative regulatory Tyr-292 of ZAP-70, thereby counteracting inactivation of ZAP-70. Expression of low levels of LMPTP resulted in increased ZAP-70 phosphorylation, presumably at the activating Tyr-493 and other sites, increased kinase activity, and augmented downstream signaling to the mitogen-activated protein kinase pathway. The ZAP-70 Y292F mutant was not affected by LMPTP. Our results indicate that LMPTP, like CD45, dephosphorylates a negative regulatory tyrosine site in a protein-tyrosine kinase and thereby strengthens T cell receptor signaling.     

Distinct tyrosine phosphorylation sites in ZAP-70 mediate activation and negative regulation of antigen receptor function.

Biochemical and genetic evidence has implicated two families of protein tyrosine kinases (PTKs), the Src- and Syk-PTKs, in T- and B-cell antigen receptor signaling. ZAP-70 is a member of the Syk-PTKs that associates with the T-cell antigen receptor and undergoes tyrosine phosphorylation following receptor activation. Three tyrosine residues, Tyr-292, -492, and -493, have been identified as sites of phosphorylation following T-cell antigen receptor engagement. Utilizing ZAP-70- and Syk-deficient lymphocytes (Syk-DT40 cells), we provide biochemical and functional evidence that heterologous trans-phosphorylation of Tyr-493 by a Src-PTK is required for antigen receptor-mediated activation of both the calcium and ras pathways. In contrast, cells expressing mutations at Tyr-292 or -492 demonstrate hyperactive T- and B-cell antigen receptor phenotypes. Thus, phosphorylation of ZAP-70 mediates both activation and inactivation of antigen receptor signaling.     

ERK Positive Feedback Regulates a Widespread Network of Tyrosine Phosphorylation Sites across Canonical T Cell Signaling and Actin Cytoskeletal Proteins in Jurkat T Cells

Competing positive and negative signaling feedback pathways play a critical role in tuning the sensitivity of T cell receptor activation by creating an ultrasensitive, bistable switch to selectively enhance responses to foreign ligands while suppressing signals from self peptides. In response to T cell receptor agonist engagement, ERK is activated to positively regulate T cell receptor signaling through phosphorylation of Ser⁵⁹ Lck. To obtain a wide-scale view of the role of ERK in propagating T cell receptor signaling, a quantitative phosphoproteomic analysis of 322 tyrosine phosphorylation sites by mass spectrometry was performed on the human Jurkat T cell line in the presence of U0126, an inhibitor of ERK activation. Relative to controls, U0126-treated cells showed constitutive decreases in phosphorylation through a T cell receptor stimulation time course on tyrosine residues found on upstream signaling proteins (CD3 chains, Lck, ZAP-70), as well as downstream signaling proteins (VAV1, PLCγ1, Itk, NCK1). Additional constitutive decreases in phosphorylation were found on the majority of identified proteins implicated in the regulation of actin cytoskeleton pathway. Although the majority of identified sites on T cell receptor signaling proteins showed decreases in phosphorylation, Tyr⁵⁹⁸ of ZAP-70 showed elevated phosphorylation in response to U0126 treatment, suggesting differential regulation of this site via ERK feedback. These findings shed new light on ERK’s role in positive feedback in T cell receptor signaling and reveal novel signaling events that are regulated by this kinase, which may fine tune T cell receptor activation.     

Analysis of recombinant DNA-derived glycoproteins via high-performance capillary electrophoresis coupled with off-line matrix-assisted laser desorption ionization time-of-flight mass spectrometry

This paper describes the analysis of glycoform populations of the glycoproteins ovalbumin and Desmodus salivary plasminogen activator (DSPA α1) by a combination of capillary electrophoresis (CE) and off-line matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Ovalbumin has a single N-linked glycosylation site and DSPA α1 has six sites for potential glycosylation, 2 N-linked and four O-linked. The conditions used for the electrophoretic separation of ovalbumin include a borate buffer system, together with a diamine additive such as 1,4-diaminobutane (DAB). An electropherogram of DSPA glycoforms could be obtained at pH 3.0 (phosphate buffer) using a bovine serum albumin (BSA) coated capillary. Fraction collection was performed by controlled application of pressure [5000 Pa (50 mbar)] for zone elution and MALDI-TOF-MS was performed on samples prepared by a 1:1 dilution with the UV absorbing matrix sinapinic acid. Both electrophoretic separations were successfully characterized by good quality mass spectra and distinct mass trends were observed for the collected fractions. It is likely that each of the collected fractions are still mixtures of glycoforms and explanation of relative mobilities or masses of different fractions is not possible at this stage. The ability to perform rapid off-line MALDI-TOF-MS of fractions from complex electropherograms will be a powerful tool to demonstrate product consistency in the manufacture of glycoprotein pharmaceuticals.     

Laser-induced dissociation of phosphorylated peptides using matrix assisted laser desorption/ionization tandem time-of-flight mass spectrometry

Reversible phosphorylation is one of the most important posttranslational modifications of cellular proteins. Mass spectrometry is a widely used technique in the characterization of phosphorylated proteins and peptides. Similar to nonmodified peptides, sequence information for phosphopeptides digested from proteins can be obtained by tandem mass analysis using either electrospray ionization or matrix assisted laser desorption/ionization (MALDI) mass spectrometry. However, the facile loss of neutral phosphoric acid (H₃PO₄) or HPO₃ from precursor ions and fragment ions hampers the precise determination of phosphorylation site, particularly if more than one potential phosphorylation site or concensus sequence is present in a given tryptic peptide. Here, we investigated the fragmentation of phosphorylated peptides under laser-induced dissociation (LID) using a MALDI-time-of-flight mass spectrometer with a curved-field reflectron. Our data demonstrated that intact fragments bearing phosphorylated residues were produced from all tested peptides that contain at least one and up to four phosphorylation sites at serine, threonine, or tyrosine residues. In addition, the LID of phosphopeptides derivatized by N-terminal sulfonation yields simplified MS/MS spectra, suggesting the combination of these two types of spectra could provide an effective approach to the characterization of proteins modified by phosphorylation.     

Tracking and quantification of P-labeled phosphopeptides in liquid chromatography matrix-assisted laser desorption/ionization mass spectrometry

Phosphoamino acid modifications on substrate proteins are critical components of protein kinase signaling pathways. Thus, diverse methodologies have been developed and applied to identify the sites of phosphorylated amino acids within proteins. Despite significant progress in the field, even the determination of phosphorylated residues in a given highly purified protein is not a matter of routine and can be difficult and time-consuming. Here we present a practicable approach that integrates into a liquid chromatography matrix-assisted laser desorption/ionization mass spectrometry (LC–MALDI MS) workflow and allows localization and quantification of phosphorylated peptides on the MALDI target plate prior to MS analysis. Tryptic digests of radiolabeled proteins are fractionated by reversed-phase LC directly onto disposable MALDI target plates, followed by autoradiographic imaging. Visualization of the radiolabel enables focused analysis of selected spots, thereby accelerating the process of phosphorylation site mapping by decreasing the number of spectra to be acquired. Moreover, absolute quantification of the phosphorylated peptides is permitted by the use of appropriate standards. Finally, the manual sample handling is minimal, and consequently the risk of adsorptive sample loss is very low. Application of the procedure allowed the targeted identification of six novel autophosphorylation sites of AMP-activated protein kinase (AMPK) and displayed additional unknown phosphorylated peptide species not amenable to detection by MS. Furthermore, autoradiography revealed topologically inhomogeneous distribution of phosphorylated peptides within individual spots. However, accurate analysis of defined areas within single spots suggests that, rather than such quantitative differences, mainly the manner of matrix crystallization significantly affects ionization of phosphopeptides.     

Non-degradative Ubiquitination of Protein Kinases

Growing evidence supports other regulatory roles for protein ubiquitination in addition to serving as a tag for proteasomal degradation. In contrast to other common post-translational modifications, such as phosphorylation, little is known about how non-degradative ubiquitination modulates protein structure, dynamics, and function. Due to the wealth of knowledge concerning protein kinase structure and regulation, we examined kinase ubiquitination using ubiquitin remnant immunoaffinity enrichment and quantitative mass spectrometry to identify ubiquitinated kinases and the sites of ubiquitination in Jurkat and HEK293 cells. We find that, unlike phosphorylation, ubiquitination most commonly occurs in structured domains, and on the kinase domain, ubiquitination is concentrated in regions known to be important for regulating activity. We hypothesized that ubiquitination, like other post-translational modifications, may alter the conformational equilibrium of the modified protein. We chose one human kinase, ZAP-70, to simulate using molecular dynamics with and without a monoubiquitin modification. In Jurkat cells, ZAP-70 is ubiquitinated at several sites that are not sensitive to proteasome inhibition and thus may have other regulatory roles. Our simulations show that ubiquitination influences the conformational ensemble of ZAP-70 in a site-dependent manner. When monoubiquitinated at K377, near the C-helix, the active conformation of the ZAP-70 C-helix is disrupted. In contrast, when monoubiquitinated at K476, near the kinase hinge region, an active-like ZAP-70 C-helix conformation is stabilized. These results lead to testable hypotheses that ubiquitination directly modulates kinase activity, and that ubiquitination is likely to alter structure, dynamics, and function in other protein classes as well.     

Interdomain B in ZAP-70 Regulates but Is Not Required for ZAP-70 Signaling Function in Lymphocytes

The protein tyrosine kinase ZAP-70 plays an important role in T-cell activation and development. After T-cell receptor stimulation, ZAP-70 associates with the receptor and is phosphorylated on many tyrosines, including Y292, Y315, and Y319 within interdomain B. Previously, we demonstrated that Y292 negatively regulates ZAP-70 function and that Y315 positively regulates ZAP-70 function by interacting with Vav. Recent studies have suggested that Y319 also positively regulate ZAP-70 function. Paradoxically, removal of interdomain B (to create the construct designated Δ), containing the Y292, Y315, and Y319 sites, did not eliminate the ability of ZAP-70 to induce multiple gene reporters in Syk-deficient DT-40 B cells and ZAP-70/Syk-deficient Jurkat cells. Here we show that Δ still utilizes the same pathways as wild-type ZAP-70 to mediate NF-AT induction. This is manifested by the ability of Δ to restore induction of calcium fluxes and mitogen-activated protein kinase activation and by the ability of dominant negative Ras and FK506 to block the induction of NF-AT activity mediated by Δ. Biochemically we show that the stimulated tyrosine phosphorylation of Vav, Shc, and ZAP-70 itself is diminished, whereas that of Slp-76 is increased in cells reconstituted with Δ. Deletion of interdomain B did not affect the ability of ZAP-70 to bind to the receptor. The in vitro kinase activity of ZAP-70 lacking interdomain B was markedly reduced, but the kinase activity was still required for the protein’s in vivo activity. Based on these data, we concluded that interdomain B regulates but is not required for ZAP-70 signaling function leading to cellular responses.     

Comparison of peptides in the phloem sap of flowering and non-flowering Perilla and lupine plants using microbore HPLC followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Physiological evidence indicates that flower formation is hormonally controlled. The floral stimulus, or florigen, is formed in the leaves as a response to an inductive photoperiod and translocated through the phloem to the apical meristem. However, because of difficulties in obtaining and analyzing phloem sap and the lack of a bioassay, the chemical nature of this stimulus is one of the major unsolved problems in plant biology. A combination of microbore high-performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was used to compare the contents of the phloem sap from flowering and non-flowering plants. Instead of using one- or two-dimensional gel electrophoresis, microbore HPLC separations allowed us to detect proteins/peptides that were very small and present at very low levels. We detected more than 100 components in the phloem sap of Perilla ocymoides L. and Lupinus albusL. Sequences for 16 peptides in a mass range from 1 to 9 kDa were obtained. Two of these could be identified, 11 showed similarity to known or deduced protein sequences, and three showed no similarity to any known protein or translated gene sequence. Four of these peptides were specific to, modified, or increased in plants that were flowering, indicating their possible role in flower induction. The sequences of these peptides showed similarities to two purine permeases, a protein with similarity to protein kinases, and a protein with no similarities to any known protein.     

TiO(2)-based phosphoproteomic analysis of the plasma membrane and the effects of phosphatase inhibitor treatment

Phosphorylation of plasma membrane proteins frequently initiates signal transduction pathways or attenuate plasma membrane transport processes. Because of the low abundance and hydrophobic features of many plasma membrane proteins and the low stoichiometry of protein phosphorylation, studies of the plasma membrane phosphoproteome are challenging. We present an optimized analytical strategy for plasma membrane phosphoproteomics that combines efficient plasma membrane protein preparation with TiO(2)-based phosphopeptide enrichment and high-performance mass spectrometry for phosphopeptide sequencing. We used sucrose centrifugation in combination with sodium carbonate extraction to achieve efficient and reproducible purification of low microgram levels of plasma membrane proteins from human mesenchymal stem cells (hMSCs, 10(7) cells), achieving more than 70% yield of membrane proteins. Phosphopeptide enrichment by titanium dioxide chromatography followed by capillary liquid chromatography-tandem mass spectrometry allowed us to assign 703 unique phosphorylation sites in 376 phosphoproteins. Our experiments revealed that treatment of cell cultures with three different types of protein phosphatase inhibitors produces distinct phosphopeptide populations and an increase of 10-40% of the number of detected and sequenced phosphoserine, phosphothreonine and phosphotyrosine containing peptides. In summary, our analytical strategy enables functional phosphoproteomic analysis of stem cell differentiation and cell surface biomarker discovery using very low amounts of starting material.     

High-sensitivity determination of tyrosine-phosphorylated peptides by on-line enzyme reactor and electrospray ionization mass spectrometry.

We describe a simple, fast, sensitive, and nonisotopic bioanalytical technique for the detection of tyrosine-phosphorylated peptides and the determination of sites of protein tyrosine phosphorylation. The technique employs a protein tyrosine phosphatase micro enzyme reactor coupled on-line to either capillary electrophoresis or liquid chromatography and electrospray ionization mass spectrometry instruments. The micro enzyme reactor was constructed by immobilizing genetically engineered, metabolically biotinylated human protein tyrosine phosphatase beta onto the inner surface of a small piece of a 50-microns inner diameter, 360-microns outer diameter fused silica capillary or by immobilization of the phosphatase onto 40-90-microns avidin-activated resins. By coupling these reactors directly to either a capillary electrophoresis column or a liquid chromatography column, we were able to rapidly perform enzymatic dephosphorylation and separation of the reaction products. Detection and identification of the components of the reaction mixture exiting these reactors were done by mass analysis with an on-line electrospray ionization mass spectrometer. Tyrosine-phosphorylated peptides, even if present in a complex peptide mixture, were identified by subtractive analysis of peptide patterns generated with or without phosphatase treatment. Two criteria, namely a phosphatase-induced change in hydropathy and charge, respectively, and a change in molecular mass by 80 Da, were used jointly to identify phosphopeptides. We demonstrate that, with this technique, low picomole amounts of a tyrosine-phosphorylated peptide can be detected in a complex peptide mixture generated by proteolysis of a protein and that even higher sensitivities can be realized if more sensitive detection systems are applied.     

Determination of the Substrate Specificity of Protein-tyrosine Phosphatase TULA-2 and Identification of Syk as a TULA-2 Substrate

TULA-1 (UBASH3A/STS-2) and TULA-2 (p70/STS-1) represent a novel class of protein-tyrosine phosphatases. Previous studies suggest that TULA-2 is sequence-selective toward phosphotyrosyl (Tyr(P)) peptides. In this work the substrate specificity of TULA-1 and -2 was systematically evaluated by screening a combinatorial Tyr(P) peptide library. Although TULA-1 showed no detectable activity toward any of the Tyr(P) peptides in the library, TULA-2 recognizes two distinct classes of Tyr(P) substrates. On the N-terminal side of Tyr(P), the class I substrates contain a proline at the Tyr(P)−1 position, a hydrophilic residue at the Tyr(P)−2 position, and aromatic hydrophobic residues at positions Tyr(P)−3 and beyond. The class II substrates typically contain two or more acidic residues, especially at Tyr(P)−1 to Tyr(P)−3 positions, and aromatic hydrophobic residues at other positions. At the C-terminal side of Tyr(P), TULA-2 generally prefers acidic and aromatic residues. The library screening results were confirmed by kinetic analysis of representative peptides selected from the library as well as Tyr(P) peptides derived from various Tyr(P) proteins. TULA-2 is highly active toward peptides corresponding to the Tyr(P)-323 and Tyr(P)-352 sites of Syk, and the Tyr(P)-397 site of focal adhesion kinase and has lower activity toward other Tyr(P) sites in these proteins. In glycoprotein VI-stimulated platelets, knock-out of the TULA-2 gene significantly increased the phosphorylation level of Syk at Tyr-323 and Tyr-352 sites and to a lesser degree at the Tyr-525/526 sites. These results suggest that Syk is a bona fide TULA-2 substrate in platelets.     

Low-mass proteome analysis based on liquid chromatography fractionation,nanoliter protein concentration/digestion,and microspot matrix-assisted laser desorption ionization mass spectrometry

HPLC fractionation combined with mass spectrometry can become a powerful tool for analyzing the proteome in the mass range below 15 kDa where efficient protein separation by gel electrophoresis can be difficult. For sensitive and high-resolution separation of the low-mass proteome, the use of analytical rather than preparative HPLC columns is preferred. However, individual fractions collected by a conventional HPLC separation usually contain a small amount of proteins whose concentrations may not be sufficiently high for subsequent enzyme digestion and protein identification by mass spectrometry. In this work, we present a high sensitivity nanoliter sample handling technique to analyze proteins fractionated by HPLC. In this technique, an individual HPLC fraction in hundreds of microliter volume is pre-concentrated to several microliters. About 700 pl of the pre-concentrated fraction is then drawn into a 20-microm I.D. capillary and dried in a small region near the capillary's entrance. This process can be repeated many times to concentrate a sufficient amount of protein to the small region of the capillary. After protein concentration, protein digestion is achieved by drawing 1 nl of chemical or enzymatic reagent into the capillary and placing it in the same region where the dried protein sits. The resulting peptides are then deposited onto a microspot in a MALDI probe for mass analysis. The performance of this technique is demonstrated with the use of a standard protein solution. This technique is applied to the identification of low-mass proteins separated by HPLC from a complex mixture of an E. coli extract.     

A decade of high-resolution liquid chromatography of nucleic acids on styrene-divinylbenzene copolymers

The introduction of alkylated, nonporous poly-(styrene-divinylbenzene) microparticles in 1992 enabled the subsequent development of denaturing HPLC that has emerged as the most sensitive screening method for mutations to date. Denaturing HPLC has provided unprecedented insight into human origins and prehistoric migrations, accelerated the cloning of genes involved in mono- and polygenic traits, and facilitated the mutational analysis of more than a hundred candidate genes of human disease. A significant step toward increased sample-throughput and information content was accomplished by the recent introduction of monolithic poly(styrene-divinylbenzene) capillary columns. They have enabled the construction of capillary arrays amenable to multiplex analysis of fluorescent dye-labeled nucleic acids by laser-induced fluorescence detection. Hyphenation of denaturing HPLC with electrospray ionization mass spectrometry, on the other hand, has allowed the direct elucidation of the chemical nature of DNA variation and determination of phase of multiple alleles on a chromosome.     

Protein cross-links: universal isolation and characterization by isotopic derivatization and electrospray ionization mass spectrometry.

A general method of unequivocally identifying and obtaining sequence information on cross-linked peptides derived by proteolytic digestion of cross-linked proteins has been developed. The method is based on isotopic labeling of alpha-amino groups with 2, 4-dinitrofluorobenzene (DNFB) coupled with electrospray ionization mass spectrometry. Proteins containing covalent cross-link(s) are reductively methylated to convert lysine residues to dimethyl lysine. The methylated protein is partially hydrolyzed and the liberated alpha-amino termini are derivatized with an equimolar mixture of DNFB and [(2)H(3)]DNFB. Dinitrophenyl (DNP)-labeled peptides may be fractionated into mono- and bis-DNP pools by chromatography on phenyl media. The bis-DNP peptides are further separated by reverse-phase HPLC and analyzed by electrospray ionization mass spectrometry. The molecular ions of cross-linked peptides are unambiguously identified as 1:2:1 triplets in the mass spectrum resulting from the binomial distribution of isotopic label in the bis-DNP derivative. Sequence information can be elucidated from the unique product ion patterns which are generated from in-source fragmentation at an elevated cone voltage. Analysis of the disulfide cross-linked peptide (VTCG)(2) was undertaken as a proof of concept and the generality of the method was demonstrated by isolating and sequencing the isopeptide bond of polyubiquitin.     

Differential and multiple binding of signal transducing molecules to the ITAMs of the TCR-ζ chain

A biotin-streptavidin-based technique was developed for high affinity, unidirectional, and specific immobilization of synthetic peptides to a solid phase. Biotinylated 23-mer carboxamide peptides corresponding to the three immunoreceptor tyrosine-based activation motifs (ITAMs) of the T cell antigen receptor associated ζ-chain (TCR-ζ) in their bis-, mono-, or unphosphorylated forms were used to study the binding of cellular proteins from human Jurkat T cells to these signal transduction motifs. The protein tyrosine kinase ZAP-70 bound specifically to all bisphosphorylated peptides but not to the mono- or unphosphorylated peptides. In contrast, Shc, phosphatidylinositol 3-kinase (P13K), Grb2, and Ras-GTPase activating protein (GAP) bound with different affinities to the bis- or monophosphorylated peptides, while the Src family protein tyrosine kinase (PTK) Fyn did not bind specifically to any of the tested peptides. The different preferences of the studied signaling molecules for distinct ITAMs, and in particular the binding of some of them preferentially to monophosphorylated peptides, suggests that the TCR-ζ may bind multiple signaling molecules with each ITAM binding a unique set of such molecules. In addition, partial phosphorylation of the ITAMs may result in recruitment of different proteins compared to double phosphorylation. This may be crucial for coupling of the TCR to various effector functions under different conditions of receptor triggering. © 1996 Wiley-Liss, Inc.     

Tandem mass spectrometric method for definitive localization of phosphorylation sites using bromine signature

Determination of the phosphorylation site in peptides by conventional tandem mass spectrometry is subject to ambiguity due to the neutral loss of the phosphate groups, especially in multiphosphorylated peptides. To prevent the neutral loss, the phosphate groups in phosphoserine or phosphothreonine peptides were replaced by p-bromobenzyl mercaptan via β-elimination and Michael addition. The unique isotopic signature of the Br introduced facilitated definitive localization of phosphorylation sites in multiphosphorylated peptides with highly adjacent serine or threonine residues. This method could be used to confirm phosphorylation sites determined by conventional tandem mass spectrometry after phosphopeptide enrichment.     

Mapping sites of O-GlcNAc modification using affinity tags for serine and threonine post-translational modifications

Identifying sites of post-translational modifications on proteins is a major challenge in proteomics. O-Linked beta-N-acetylglucosamine (O-GlcNAc) is a dynamic nucleocytoplasmic modification more analogous to phosphorylation than to classical complex O-glycosylation. We describe a mass spectrometry-based method for the identification of sites modified by O-GlcNAc that relies on mild beta-elimination followed by Michael addition with dithiothreitol (BEMAD). Using synthetic peptides, we also show that biotin pentylamine can replace dithiothreitol as the nucleophile. The modified peptides can be efficiently enriched by affinity chromatography, and the sites can be mapped using tandem mass spectrometry. This same methodology can be applied to mapping sites of serine and threonine phosphorylation, and we provide a strategy that uses modification-specific antibodies and enzymes to discriminate between the two post-translational modifications. The BEMAD methodology was validated by mapping three previously identified O-GlcNAc sites, as well as three novel sites, on Synapsin I purified from rat brain. BEMAD was then used on a purified nuclear pore complex preparation to map novel sites of O-GlcNAc modification on the Lamin B receptor and the nucleoporin Nup155. This method is amenable for performing quantitative mass spectrometry and can also be adapted to quantify cysteine residues. In addition, our studies emphasize the importance of distinguishing between O-phosphate versus O-GlcNAc when mapping sites of serine and threonine post-translational modification using beta-elimination/Michael addition methods.     

Mass spectrometric analysis of the kinetics of in vivo rhodopsin phosphorylation

On stimulation, rhodopsin, the light-sensing protein in the rod cells of the retina, is phosphorylated at several sites on its C terminus as the first step in deactivation. We have developed a mass spectrometry-based method to quantify the kinetics of phosphorylation at each site in vivo. After exposing either a freshly dissected mouse retina or the eye of an anesthetized mouse to a flash of light, phosphorylation and dephosphorylation reactions are terminated by rapidly homogenizing the retina or enucleated eye in 8 M urea. The C-terminal peptide containing all known phosphorylation sites is cleaved from rhodopsin, partially purified by ultracentrifugation, and analyzed by liquid chromatography coupled with mass spectrometry (LCMS). The mass spectrometer responds linearly to the peptide from 10 fmole to 100 pmole. The relative sensitivity to peptides with zero to five phosphates was determined using purified phosphopeptide standards. High pressure liquid chromatography (HPLC) coupled with tandem mass spectrometry (LCMS/MS) was used to distinguish the three primary sites of phosphorylation, Ser 334, Ser 338, and Ser 343. Peptides monophosphorylated on Ser 334 were separable from those monophosphorylated on Ser 338 and Ser 343 by reversed-phase HPLC. Although peptides monophosphorylated at Ser 338 and Ser 343 normally coelute, the relative amounts of each species in the single peak could be determined by monitoring the ratio of specific daughter ions characteristic of each peptide. Doubly phosphorylated rhodopsin peptides with different sites of phosphorylation also were distinguished by LCMS/MS. The sensitivity of these methods was evaluated by using them to measure rhodopsin phosphorylation stimulated either by light flashes or by continuous illumination over a range of intensities.