Phosphorylation Degrees Research Articles

Separation of multiphosphorylated peptide isomers by CZE

A separation of mono, doubly and triply phosphorylated isomers was developed with CZE with an aqueous electrolyte containing 3.9 mol/L formic acid and 30% v/v trifluoroethanol. Thus a mixture of ten phosphopeptides corresponding to the human tau sequence 226-240 was separated within 70 min. Although peptides with different phosphorylation degrees, i.e. 0-3 phosphate groups, were well separated, some of the phosphopeptide isomers containing one or two phosphate groups were only partially separated. The electrolyte system is compatible with both MALDI- and ESI-MS, allowing a direct coupling, and thus could have some interesting applications in proteomics.

Mass spectrometric quantification of the relative amounts of C6 and C3 position phosphorylated glucosyl residues in starch

The quantification of phosphate bound to the C6 and C3 positions of glucose residues in starch has received increasing interest since the importance of starch phosphorylation for plant metabolism was discovered. The method described here is based on the observation that the isobaric compounds glucose-6-phosphate (Glc6P) and glucose-3-phosphate (Glc3P) exhibit significantly different fragmentation patterns in negative ion electrospray tandem mass spectrometry (MS/MS). A simple experiment involving collision-induced dissociation (CID) MS 2 spectra of the sample and the two reference substances Glc3P and Glc6P permitted the quantification of the relative amounts of the two compounds in monosaccharide mixtures generated by acid hydrolysis of starch. The method was tested on well-characterized potato tuber starch. The results are consistent with those obtained by NMR analysis. In contrast to NMR, however, the presented method is fast and can be performed on less than 1 mg of starch. Starch samples of other origins exhibiting a variety of phosphorylation degrees were analyzed to assess the sensitivity and robustness of the method.

Modulation of erythrocyte deformability by PKC activity

The interactions between membrane, peripheral and cytoskeleton proteins are responsible for the maintenance of erythrocyte deformability (EEI) and some of these interactions are modulated by PKC activity. Protein band 3 of the erythrocyte membrane is phosphorylated by phosphotyrosine kinases (PTK) and dephosphorylated by phosphotyrosine phosphatase (PTP). It was previously described by us a signal transduction mechanism that describes a possible pathway connecting an erythrocyte external membrane protein, acetylcholinesterase (AChE), with protein band 3. So how does PKC activity modulate EEI when protein band 3 is phosphorylated or dephosphorylated in absence or presence of AChE effectors?To answer this we used phorbol 12-myristate 13-acetate (PMA) as an activator and chelerythrine chloride as inhibitor of PKC and also band 3 modulators of band 3 phosphorylation degree, in presence and absence of AChE effectors in order to measure in whole blood samples EEI. Our results showed that erythrocyte deformability was significantly (i) decreased by inhibition of PKC, in absence and presence of AChE inhibitor velnacrine (ii) increased with PMA in absence and presence of ACh and (iii) decreased in presence of calpeptin in absence and presence of either chelerythrine or PMA. These results establish dependence between cytoskeleton proteins, PKC activity, band 3 phosphorylation degrees and EEI. Better understanding of those proteins interactions on transduction mechanisms might trigger possible targets for drug action that would modulate EEI.

The role of sulfur and sulfur isotope dilution analysis in quantitative protein analysis

The element sulfur is almost omnipresent in all natural proteomes and plays a key role in protein quantification. Incorporated in the amino acids cysteine and methionine, it has been served as target for many protein-labeling reactions in classic quantitative proteomic approaches based on electrospray or MALDI mass spectrometry. This critical review discusses the potential and limitations of sulfur isotope dilution analysis (IDA) by inductively coupled plasma-mass spectrometry (ICP-MS) for absolute protein quantification. The development of this approach was made possible due to the improved sensitivity and accuracy of sulfur isotope ratio measurement by ICP-MS in recent years. The unique feature of ICP-MS, compound-independent ionization, enables compound (species)-unspecific sulfur IDA. This has the main advantage that only one generic sulfur standard (i.e., one isotopically labeled sulfur spike) is required to quantify each peptide or protein in a sample provided that they are completely separated in chromatography or electrophoresis and that their identities are known. The principles of this approach are illustrated with selected examples from the literature. The discussion includes also related fields of P/S and metal/S ratio measurements for the determination of phosphorylation degrees of proteins and stoichiometries in metalloproteins, respectively. Emerging new areas and future trends such as protein derivatization with metal tags for improved sensitivity of protein detection in ICP-MS are discussed.

Determination of phosphorylation degrees in caseins by on-line gel electrophoresis coupled to ICP-SFMS

The determination of the phosphorylation degree of proteins is essential in many biochemical fields. Besides mass spectrometric techniques elucidating the protein structure, ICP-MS has entered this research area as it provides excellent quantification capabilities. In this work the separation of the proteins has been carried out by gel electrophoresis on-line coupled to an ICP equipped with a double-focusing sector field-MS (ELEMENT 2). Based on the simultaneous detection of 32S+ and 31P+, this work presents an alternative approach to determining the phosphorylation degree of caseins. For the example of α- and β-casein the results obtained were in excellent agreement with the expected values, and therefore the described method might be very useful and serve as an alternative in the analysis of phosphorylated proteins.

Effects of insulin receptor substrate-1 and its serine phosphorylation and tyrosine phosphorylation on insulin resistance in skeletal muscle cells in the state of sepsis: experiment with rats

To investigate the effects of insulin receptor substrate (IRS)-1 and its serine (Ser)(307) phosphorylation and tyrosine (Tyr) phosphorylation on insulin resistance in skeletal muscle cells in the state of sepsis. 120 SD rats were randomly divided into 3 groups: 10% group, with 10% of the total cecal length ligated and punctured without use of antibiotic so as to make sepsis model; 30% group, with 30% of the total cecal length ligated and punctured; and control group, undergoing sham operation. Fasting venous blood samples were collected before the operation to detect the fasting plasma glucose (FPG). 0, 8, 16, 24, and 48 hours after the operation 8 rats in each group underwent fasting of food and without fasting of water for 8 hours, i.e., until the 8 th, 16 th, 24 th, 48 th, and 72 nd hours after the operation. Then the rats underwent anesthesia, with blood sample from the vena cava and specimen of gastrocnemius of the hind leg collected, and then killed. The levels of FPG, fasting plasma insulin (FINS), tumor necrosis factor (TNF)-alpha, and interleukin (IL)-6 were measured. HOMA method was used to calculate the insulin resistance index (Ig-IRI). Immunohistochemistry was used to quantitatively examine the IRS-1 protein and its Ser(307) phosphorylation and Tyr phosphorylation in the gastrocnemius. Western blotting and immunoprecipitation were used to semi-quantitatively examine the changes in contents of IRS-1 and its Ser(307) phosphorylation and Tyr phosphorylation in the gastrocnemius respectively. The survival rates at different time points of the control group were all 100%, all significantly higher than those of the other 2 groups (all P < 0.01), and those of the 10% group were all significantly higher than those of the 30% group (all P < 0.01). The levels of TNF-alpha and IL-6 of the 10% and 30% groups at different time points were all significantly higher than those of the control group (all P < 0.01), and those of the 30% group were all significantly higher than those of the 10% group (all P < 0.01). The FPG, FINS, and IgIRI were not significantly different among the 3 groups before the operation, and those of the 10% and 30% groups at different time points after operation were all significantly higher than those of the control group (all P < 0.01) and peaked 8 h after the operation, with those of the 30% group all significantly higher than those of the 10% group (all P < 0.01). The degree of increase of FINS was remarkably higher than that of FPG. IRS-1 was positive and located in the cytoplasm of the gastrocnemius cells in both the control and 30% groups; IRS-1 Tyr phosphorylation was positive in the control group and sporadic positive in the 30% group. IRS-1 Ser(307) was negative in the control group and strong positive in the 30% group. Semi-quantitative examination showed that the IRS-1 level at different time points after operation of the 30% group were not significantly different from those of the control group (all P > 0.05), and IRS-1 Tyr phosphorylation degrees at different time points of the 30% group were all significantly lower than those of the control group (all P < 0.01), and the IRS-1 Ser(307) phosphorylation at different time points of the 30% group were all significantly higher than those of the control group (all P < 0.01). Spearman correlation analysis showed that IgIRI was significantly negatively correlated with IRS-1 Tyr phosphorylation (r = -0.957, P < 0.01), and significantly positively correlated with IRS-1 Ser(307) phosphorylation (r = -0.955, P < 0.01). Under the status of sepsis the IRS-1 content in the skeletal muscle cells is unchanged, the level of IRS-1 Tyr phosphorylation level is decreased, and the IRS-1 Ser phosphorylation is increased. The degrees of such changes are closely related with the degree of insulin resistance.

Double-Hydrophilic Block Copolymers with Monophosphate Ester Moieties as Crystal Growth Modifiers of CaCO3

The synthesis of double-hydrophilic block copolymers with a poly(ethylene glycol) block (PEG) and a block with pendant monophosphate ester groups based on a hydroxylated polybutadiene block (poly[2-(2-hydroxy ethyl)ethylene] (PHEE) with variable degrees of phosphate substitution (up to 40%) is described. It is shown that these block copolymers are very efficient scale inhibitors for CaCO3. The efficiency of these polymers is compared with block copolymers with an ionic block based on phosphorylated polyglycidol (PGL) with phosphorylation degrees up to 100%. The phosphorylated polyglycidols were also used to modify the morphology of CaCO3 crystals. Instead of the typical rhombohedral calcite single crystals, superstructures of nanometer-sized particles are formed in the presence of these block copolymers when two different techniques were used: the fast double-jet technique and the slow Kitano-method. In the double-jet method, only spherical superstructures are obtained, whereas for the slower growth region covered by the Kitano method, complex cone-like or flower-like superstructures are formed.

Stepwise subunit interaction changes by mono- and bisphosphorylation of cardiac troponin I.

Four phosphorylation degrees of cardiac troponin I (cTnI) have been characterized, namely, a dephospho, a bisphospho, and two monophospho states. Here we describe for the first time a role of the monophosphorylated forms. We have investigated the interaction between the cardiac troponin subunits dependent on the phosphorylation state of cTnI by surface plasmon resonance (SPR) spectroscopy. The monophosphorylated forms were generated by mutating each of the two serine residues, located in human cTnI at positions 22 and 23, to alanine. Association and dissociation rate constants of binary (cTnI-cTnT and cTnI-cTnC) and ternary (cTnI/cTnC complex-cTnT) complexes were determined. Mono- and consecutive bisphosphorylation of cTnI gradually reduces the affinity to cTnC and cTnT by lowering the association rate constants; the dissociation rate constants remain unchanged. Phosphorylation also affects formation of the ternary complexes; however, in this instance, association rate constants are constant, and dissociation rate constants are enhanced. A model of cardiac troponin is presented describing an induction of distinct conformational changes by mono- and bisphosphorylation of cTnI.

Duration and amplitude of the light-induced cGMP hydrolysis in vertebrate photoreceptors are regulated by multiple phosphorylation of rhodopsin and by arrestin binding.

The duration and amplitude of the light-induced cGMP hydrolysis in bovine rod outer segments were investigated using purified rhodopsin in nine different states of phosphorylation in a reconstituted system. Effects of varying amounts of arrestin at all states of rhodopsin phosphorylation were measured. The findings were the following: (1) At low bleaching levels, the activity of phosphodigesterase (PDE) depends strongly on the phosphorylation degree of the light-activated rhodopsin (R*), while at saturating light levels R* of all phosphorylation degrees activates PDE to the same extent. (2) The turnoff time for PDE is markedly shortened if R* is phosphorylated, independent of the number of phosphate groups incorporated into rhodopsin (P/R); i.e., the first phosphate which is bound to R* seems to be responsible for the shortened turnoff time. The lifetime of phosphorylated R* is shown to be dramatically reduced compared to that of unphosphorylated R*, as monitored by the ability of R* to activate PDE. (3) After activation with phosphorylated R*, addition of arrestin caused a further reduction of both the maximum activity and the turnoff time of PDE. Both effects were strongly dependent on (a) the phosphorylation degree of R*, (b) the concentration of arrestin, and (c) the concentration of R*. These results suggest that the light-induced phosphorylation of rhodopsin to different extents and the subsequent binding of arrestin are involved in the light adaptation and in the fine regulation of the light response in vertebrate photoreceptors.