Proteomic screening for Rho-kinase substrates by combining kinase and phosphatase inhibitors with 14-3-3ζ affinity chromatography

The small GTPase RhoA is a molecular switch in various extracellular signals. Rho-kinase/ROCK/ROK, a major effector of RhoA, regulates diverse cellular functions by phosphorylating cytoskeletal proteins, endocytic proteins, and polarity proteins. More than twenty Rho-kinase substrates have been reported, but the known substrates do not fully explain the Rho-kinase functions. Herein, we describe the comprehensive screening for Rho-kinase substrates by treating HeLa cells with Rho-kinase and phosphatase inhibitors. The cell lysates containing the phosphorylated substrates were then subjected to affinity chromatography using beads coated with 14-3-3 protein, which interacts with proteins containing phosphorylated serine or threonine residues, to enrich the phosphorylated proteins. The identities of the molecules and phosphorylation sites were determined by liquid chromatography tandem mass spectrometry (LC/MS/MS) after tryptic digestion and phosphopeptide enrichment. The phosphorylated proteins whose phosphopeptide ion peaks were suppressed by treatment with the Rho-kinase inhibitor were regarded as candidate substrates. We identified 121 proteins as candidate substrates. We also identified phosphorylation sites in Partitioning defective 3 homolog (Par-3) at Ser143 and Ser144. We found that Rho-kinase phosphorylated Par-3 at Ser144 both in vitro and in vivo. The method used in this study would be applicable and useful to identify novel substrates of other kinases.     

Nucleoside phosphorylation by phosphate minerals

In the presence of formamide, crystal phosphate minerals may act as phosphate donors to nucleosides, yielding both 5'- and, to a lesser extent, 3'-phosphorylated forms. With the mineral Libethenite the formation of 5'-AMP can be as high as 6% of the adenosine input and last for at least 10(3) h. At high concentrations, soluble non-mineral phosphate donors (KH(2)PO(4) or 5'-CMP) afford 2'- and 2':3'-cyclic AMP in addition to 5'-and 3'-AMP. The phosphate minerals analyzed were Herderite Ca[BePO(4)F], Hureaulite Mn(2+)(5)(PO(3)(OH)(2)(PO(4))(2)(H(2)O)(4), Libethenite Cu(2+)(2)(PO(4))(OH), Pyromorphite Pb(5)(PO(4))(3)Cl, Turquoise Cu(2+)Al(6)(PO(4))(4)(OH)(8)(H(2)O)(4), Fluorapatite Ca(5)(PO(4))(3)F, Hydroxylapatite Ca(5)(PO(4))(3)OH, Vivianite Fe(2+)(3)(PO(4))(2)(H(2)O)(8), Cornetite Cu(2+)(3)(PO(4))(OH)(3), Pseudomalachite Cu(2+)(5)(PO(4))(2)(OH)(4), Reichenbachite Cu(2+)(5)(PO(4))(2)(OH)(4), and Ludjibaite Cu(2+)(5)(PO(4))(2)(OH)(4)). Based on their behavior in the formamide-driven nucleoside phosphorylation reaction, these minerals can be characterized as: 1) inactive, 2) low level phosphorylating agents, or 3) active phosphorylating agents. Instances were detected (Libethenite and Hydroxylapatite) in which phosphorylation occurs on the mineral surface, followed by release of the phosphorylated compounds. Libethenite and Cornetite markedly protect the beta-glycosidic bond. Thus, activated nucleic monomers can form in a liquid non-aqueous environment in conditions compatible with the thermodynamics of polymerization, providing a solution to the standard-state Gibbs free energy change (DeltaG degrees ') problem, the major obstacle for polymerizations in the liquid phase in plausible prebiotic scenarios.     

Site-directed mutagenesis of the avian retrovirus nucleocapsid protein, pp 12, at Serine 40, the primary site of phosphorylation in vivo

The major nucleocapsid protein of avian retroviruses, pp 12, binds to single-stranded viral RNA with high affinity. Phosphorylation at Ser-40 is necessary for this binding. In order to examine the role of phosphorylation of serine 40 in the biological function of pp 12, we have introduced a series of amino acid substitutions at this position in the Rous sarcoma virus (Pr-C) protein. Substitution of threonine, alanine, or three other amino acids for Ser-40 had very little or no detectable effect on viral replication, nor did the control substitution of glycine for Ser-43, a nonphosphorylated residue. In vivo and in vitro, the Ala-40 and probably the Thr-40 substituted p 12 proteins are phosphorylated at alternative sites which are phosphorylated to a minor extent in vivo in the wild type protein. A study of the RNA binding properties of Ala-40 substituted p 12 has indicated that the protein has been stabilized in a high affinity RNA binding state which is independent of phosphorylation. The viability of the Ala-40 mutant virus indicates that this high binding affinity may be required for biological activity.     

Enrichment analysis of phosphorylated proteins as a tool for probing the phosphoproteome

The current progression from genomics to proteomics is fueled by the realization that many properties of proteins (e.g., interactions, post-translational modifications) cannot be predicted from DNA sequence. Although it has become feasible to rapidly identify proteins from crude cell extracts using mass spectrometry after two-dimensional electrophoretic separation, it can be difficult to elucidate low-abundance proteins of interest in the presence of a large excess of relatively abundant proteins. Therefore, for effective proteome analysis it becomes critical to enrich the sample to be analyzed in subfractions of interest. For example, the analysis of protein kinase substrates can be greatly enhanced by enriching the sample of phosphorylated proteins. Although enrichment of phosphotyrosine-containing proteins has been achieved through the use of high-affinity anti-phosphotyrosine antibodies, the enrichment of phosphoserine/threonine-containing proteins has not been routinely possible. Here, we describe a method for enriching phosphoserine/threonine-containing proteins from crude cell extracts, and for subsequently identifying the phosphoproteins and sites of phosphorylation. The method, which involves chemical replacement of the phosphate moieties by affinity tags, should be of widespread utility for defining signaling pathways and control mechanisms that involve phosphorylation or dephosphorylation of serine/threonine residues.     

Hydroxyapatite affinity chromatography for the highly selective enrichment of mono‐ and multi‐phosphorylated peptides in phosphoproteome analysis

The most challenging analytical task facing phosphoproteome determination requires the isolation of phosphorylated peptides from the myriad of unphosphorylated species. In the past, several strategies for phosphopeptide isolation have been proposed in combination with subsequent mass spectrometric investigations. Among these techniques, immobilized metal affinity chromatography and titanium dioxide have been recognized as the most effective. Here, we present an alternative method for the enrichment of phosphopeptides based on hydroxyapatite (HAP) chromatography. By taking advantage of the strong interaction of HAP with phosphate and calcium ions, we developed an efficient method for the selective separation and fractionation of phosphorylated peptides. The effectiveness and efficiency of recovery for this procedure was assayed using tryptic digests of standard phosphorylated protein mixtures. Based on the higher affinity of multi‐phosphorylated peptides for HAP surfaces, the introduction of a phosphate buffer gradient for stepwise peptide elution resulted in the separation of mono‐, di‐, tri‐, and multi‐phosphorylated peptides. Thus, we demonstrated that this technique is highly selective and independent of the degree of peptide phosphorylation.     

Proteomic analysis of in vivo phosphorylated synaptic proteins

In the nervous system, protein phosphorylation is an essential feature of synaptic function. Although protein phosphorylation is known to be important for many synaptic processes and in disease, little is known about global phosphorylation of synaptic proteins. Heterogeneity and low abundance make protein phosphorylation analysis difficult, particularly for mammalian tissue samples. Using a new approach, combining both protein and peptide immobilized metal affinity chromatography and mass spectrometry data acquisition strategies, we have produced the first large scale map of the mouse synapse phosphoproteome. We report over 650 phosphorylation events corresponding to 331 sites (289 have been unambiguously assigned), 92% of which are novel. These represent 79 proteins, half of which are novel phosphoproteins, and include several highly phosphorylated proteins such as MAP1B (33 sites) and Bassoon (30 sites). An additional 149 candidate phosphoproteins were identified by profiling the composition of the protein immobilized metal affinity chromatography enrichment. All major synaptic protein classes were observed, including components of important pre- and postsynaptic complexes as well as low abundance signaling proteins. Bioinformatic and in vitro phosphorylation assays of peptide arrays suggest that a small number of kinases phosphorylate many proteins and that each substrate is phosphorylated by many kinases. These data substantially increase existing knowledge of synapse protein phosphorylation and support a model where the synapse phosphoproteome is functionally organized into a highly interconnected signaling network.     

The phosphorylation of adrenal proteins in response to adrenocorticotropic hormone

The phosphorylation of rat adrenal protein components in response to adrenocorticotropin has been studied in adrenal quarters, isolated cells, and in vivo. In adrenal quarters, adrenocorticotropic hormone (ACTH)-stimulated phosphorylation or dephosphorylation of proteins was not affected by the presence of protein synthesis inhibitors despite a total inhibition of steroidogenesis. (The term dephosphorylation refers to an apparent decrease in the labeling of a particular protein with 32P at various times after the addition of ACTH. This may be due to enzymatic removal of phosphate or protein degradation or complexation of this protein with another cellular component.) Studies with isolated cell preparations identified several proteins that are phosphorylated or dephosphorylated in response to hormone. These changes in phosphorylation were also observed in adrenal quarters and correlated well with ACTH-stimulated steroidogenesis as determined by temporal analysis and dose-response studies of corticosterone production. In vivo injection of male hypophysectomized rats with [32P]phosphate and ACTH demonstrated changes in the labeling of six adrenal proteins. Many of the proteins phosphorylated in vivo were also demonstrated to be phosphorylated in both in vitro systems. Finally, the injection of a physiological dose of ACTH appeared to selectively activate the type I cAMP-dependent protein kinase within the microsomal fraction as determined by the binding of a photoaffinity-labeled reagent. These results suggest that alterations in phosphorylation of adrenal proteins in response to ACTH is proximal to or independent of the obligatory role of protein synthesis in acute steroidogenesis.     

Plant protein phosphorylation monitored by capillary liquid chromatography--element mass spectrometry

Many essential cellular functions such as growth rate, motility, and metabolic activity are linked to reversible protein phosphorylation, since they are controlled by signaling cascades based mainly on phosphorylation/dephosphorylation events. Quantification of global or site-specific protein phosphorylation is not straightforward with standard proteomic techniques. The coupling of capillary liquid chromatography (microLC) with ICP-MS (inductively coupled plasma-mass spectrometry) is a method which allows a quantitative screening of protein extracts for their phosphorus and sulfur content, and thus provides access to the protein phosphorylation degree. In extension of a recent pilot study, we analyzed protein extracts from the model organisms Arabidopsis thaliana and Chlamydomonas reinhardtii as representatives for multicellular and unicellular green photosynthetically active organisms. The results indicate that the average protein phosphorylation level of the algae C. reinhardtii is higher than that of A. thaliana. Both the average phosphorylation levels were found to be between the extreme values determined so far for prokaryotes (C. glutamicum, lowest levels) and eukaryotes (Mus musculus, highest levels). Tissue samples of A. thaliana representing different stages of plant development showed varying levels of protein phosphorylation indicating a different adjustment of the kinase/phosphatase system. We also utilized the microLC-ICP-MS technology to estimate the efficiency of a novel phosphoprotein enrichment method based on aluminum hydroxide, since the enrichment of phosphorylated species is often an essential step for their molecular characterization.     

Phosphoproteome Analysis

Protein phosphorylation is directly or indirectly involved in all important cellular events. The understanding of its regulatory role requires the discovery of the proteins involved in these processes and how, where and when protein phosphorylation takes place. Investigation of the phosphoproteome of a cell is becoming feasible today although it still represents a very difficult task especially if quantitative comparisons have to be made. Several different experimental strategies can be employed to explore phosphoproteomes and this review will cover the most important ones such as incorporation of radiolabeled phosphate into proteins, application of specific antibodies against phosphorylated residues and direct staining of phosphorylated proteins in polyacrylamide gels. Moreover, methods to enrich phosphorylated proteins such as affinity chromatography (IMAC) and immunoprecipitation as well as mass spectrometry for identification of phosphorylated peptides and phosphorylation sites are also described.     

Phosphoproteome analysis of capacitated human sperm. Evidence of tyrosine phosphorylation of a kinase-anchoring protein 3 and valosin-containing protein/p97 during capacitation

Before fertilization can occur, mammalian sperm must undergo capacitation, a process that requires a cyclic AMP-dependent increase in tyrosine phosphorylation. To identify proteins phosphorylated during capacitation, two-dimensional gel analysis coupled to anti-phosphotyrosine immunoblots and tandem mass spectrometry (MS/MS) was performed. Among the protein targets, valosin-containing protein (VCP), a homolog of the SNARE-interacting protein NSF, and two members of the A kinase-anchoring protein (AKAP) family were found to be tyrosine phosphorylated during capacitation. In addition, immobilized metal affinity chromatography was used to investigate phosphorylation sites in whole protein digests from capacitated human sperm. To increase this chromatographic selectivity for phosphopeptides, acidic residues in peptide digests were converted to their respective methyl esters before affinity chromatography. More than 60 phosphorylated sequences were then mapped by MS/MS, including precise sites of tyrosine and serine phosphorylation of the sperm tail proteins AKAP-3 and AKAP-4. Moreover, differential isotopic labeling was developed to quantify phosphorylation changes occurring during capacitation. The phosphopeptide enrichment and quantification methodology coupled to MS/MS, described here for the first time, can be employed to map and compare phosphorylation sites involved in multiple cellular processes. Although we were unable to determine the exact site of phosphorylation of VCP, we did confirm, using a cross-immunoprecipitation approach, that this protein is tyrosine phosphorylated during capacitation. Immunolocalization of VCP showed fluorescent staining in the neck of noncapacitated sperm. However, after capacitation, staining in the neck decreased, and most of the sperm showed fluorescent staining in the anterior head.     

Modification of yeast ribosomal proteins. Phosphorylation.

Two-dimensional polyacrylamide-gel electrophoretic analysis of yeast ribosomal proteins labelled in vivo with 32PO43- revealed that the proteins S2 and S10 of the 40S ribosomal subunit, and the proteins L9, L30, L44 and L45 of the 60S ribosomal subunit, are phosphorylated in vivo. Most of the phosphate groups appeared to be linked to serine residues. Teh number of phosphate groups per molecule of phosphorylated protein species ranged from 0.01 to 0.79. Since most of the phosphorylated ribosomal proteins appear to associate with the pre-ribosomal particles at a very late stage of ribosome assembly, phosphorylation is more likely to play a role in the functioning of the ribosome than in its assembly.     

From purification of large amounts of phospho-compounds (nucleotides) to enrichment of phospho-peptides using anion-exchanging resin

Through years of practice, mass spectrometry has proven to be one of the most reliable and sensitive methods for the localization of protein phosphorylation sites. Among numerous innovative methods, affinity enrichment such as immobilized metal-ion affinity chromatography followed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis appears to be the most widely chosen procedure. Here, I report a method that was originally designed for purification of large amounts of nucleotides using anion-exchanging resin but has shown the promise of enriching phosphorylated peptides. Mixtures composed of uridine monophosphate, uridine diphosphate, uridine triphosphate, and their nonphosphate compound-uridine were bottom-line separated on an anion-exchanging solid-phase extraction (SPE) column by four steps of elution with a gradient of salt concentration and pH values. The miniature form of this SPE column showed significant separation (or enrichment) of the tryptic phospho-peptides from non-phospho-peptides of the standard protein beta-casein with two steps of elution (100mM NaCl and 5% NH(4)OH). Furthermore, after utilization of this anion-exchanging-column enrichment followed by LC/MS/MS analysis on a quadrupole-tine of flight instrument, a new phosphorylation site (S191) in bovine chromogranin A was identified.     

Phosphorylation and other properties of proteins binding single-stranded DNA (SSB-proteins) from chromatin and extrachromatin fractions of Ehrlich ascites carcinoma

A comparative study of single-stranded DNA-binding proteins (SSB-proteins) isolated from chromatin and the extrachromatin fraction of Ehrlich ascites tumour cells was carried out. No differences were found either in SDS-gel electrophoretic mobility or in the single-stranded DNA-binding capacity and stimulation of the replicative synthesis of DNA. However, chromatin SSB-proteins contained 1.4-1.5 times more phosphate than extrachromatin proteins. Both preparations could be phosphorylated in vitro by protein kinase C and cAMP-dependent protein kinase, but the chromatin proteins were phosphorylated in a lesser degree. In parallel with phosphorylation the SSB-proteins displayed a higher binding affinity for ssDNA-cellulose. Phosphorylation can thus be regarded as a means of regulation of the SSB-protein function, in particular, their interaction with chromatin DNA.     

Phosphorylation by cdc2 kinase modulates DNA binding activity of high mobility group I nonhistone chromatin protein

Chromatin high mobility group protein I (HMG-I) is a mammalian nonhistone protein that has been demonstrated both in vitro and in vivo to preferentially bind to A.T-rich sequences of DNA. Recently the DNA-binding domain peptide that specifically mediates the in vitro interaction of high mobility group protein (HMG)-I with the narrow minor groove of A.T-DNA has been experimentally determined. Because of its predicted secondary structure, the binding domain peptide has been called "the A.T hook" motif. Previously we demonstrated that the A.T hook of murine HMG-I protein is specifically phosphorylated by purified mammalian cdc2 kinase in vitro and that the same site(s) are also phosphorylated in vivo in metaphase-arrested cells. We also found that the DNA binding affinity of short synthetic binding domain peptides phosphorylated in vitro by cdc2 kinase was significantly reduced compared with unphosphorylated peptides. Here we extend these findings to intact natural and recombinant HMG-I proteins. We report that the affinity of binding of full-length HMG-I proteins to A.T-rich sequences is highly dependent on ionic conditions and that phosphorylation of intact proteins by cdc2 kinase reduces their affinity of in vitro binding to A.T-DNA by about 20-fold when assayed near normal mammalian physiological salt concentrations. Furthermore, in cell synchronization studies, we demonstrated that murine HMG-I proteins are phosphorylated in vivo in a cell cycle-dependent manner on the same amino acid residues modified by purified cdc2 kinase in vitro. Together these results strongly support the assertion that HMG-I proteins are natural substrates for mammalian cdc2 kinase in vivo and that their cell cycle-dependent phosphorylation by this enzyme(s) significantly modulates their DNA binding affinity, thereby possibly altering their biological function(s).     

Ion binding properties of the dehydrin ERD14 are dependent upon phosphorylation

The ERD14 protein (early response to dehydration) is a member of the dehydrin family of proteins which accumulate in response to dehydration-related environmental stresses. Here we show the Arabidopsis dehydrin, ERD14, possesses ion binding properties. ERD14 is an in vitro substrate of casein kinase II; the phosphorylation resulting both in a shift in apparent molecular mass on SDS-PAGE gels and increased calcium binding activity. The phosphorylated protein bound significantly more calcium than the nonphosphorylated protein, with a dissociation constant of 120 microm and 2.86 mol of calcium bound per mol of protein. ERD14 is phosphorylated by extracts of cold-treated tissues, suggesting that the phosphorylation status of this protein might be modulated by cold-regulated kinases or phosphatases. Calcium binding properties of ERD14 purified from Arabidopsis extracts were comparable with phosphorylated Escherichia coli-expressed ERD14. Approximately 2 mol of phosphate were incorporated per mol of ERD14, indicating a minimum of two phosphorylation sites. Western blot analyses confirmed that threonine and serine are possible phosphorylation sites on ERD14. Utilizing matrix assisted laser desorption ionization-time of flight/mass spectrometry we identified five phosphorylated peptides that were present in both in vivo and in vitro phosphorylated ERD14. Our results suggest that the polyserine (S) domain is most likely the site of phosphorylation in ERD14 responsible for the activation of calcium binding.     

Identification and characterization of phosphorylated proteins in the human pituitary

Post-translational modifications of proteins from the human pituitary gland play an important role in the regulation of different body functions. We report on the application of a liquid chromatography-tandem mass spectrometry (MS/MS) based approach to detect and characterize phosphorylated proteins in a whole human pituitary digest. By combining an immobilized metal affinity column-based enrichment method with MS/MS conditions that favor the neutral loss of phosphoric acid from a phosphorylated precursor ion, we identified several previously undescribed phosphorylated peptides. The identified peptides were matched to the sequences of six pituitary proteins: the human growth hormone, chromogranin A, secretogranin I, 60S ribosomal protein P1 and/or P2, DnaJ homolog subfamily C member 5, and galanin. The phosphorylation sites of these important regulatory proteins were determined by MS/MS and MS(3) analysis.     

Approach to systematic analysis of serine/threonine phosphoproteome using Beta elimination and subsequent side effects: intramolecular linkage and/or racemisation

Complete analysis of the phosphorylation of serine and threonine residues directly from biological extracts is still at an early stage and will remain a challenging goal for many years. Analysis of phosphorylated proteins and identification of the phosphorylated sites in a crude biological extract is a major topic in proteomics, since phosphorylation plays a dominant role in post-translational protein modification. Beta elimination of the serine/threonine-bound phosphate by alkali action generates (methyl)dehydroalanine. The reactivity of this group susceptible of nucleophilic attacks might be used as a tool for phosphoproteome analysis. Most of the known serine/threonine kinases recognize motifs in protein targets that are rich in lysine(s) and/or arginine(s). The (methyl)dehydroalanine resulting from beta elimination of the serine/threonine-bound phosphate by alkali action is likely to react with the amino groups of these neighboring amino acids. Furthermore, the addition reaction of dehydroalanine-peptides with a nucleophilic group more likely generates diastereoisomers derivatives. The internal cyclic bonds and/or the stereoisomer peptide derivatives thus generated confer resistance to trypsin cleavage and/or constitute stop signals for exopeptidases such as carboxypeptidase. This might form the basis of a method to facilitate the systematic identification of phosphorylated peptides.     

Ligand modulates VDR-Ser/Thr protein phosphatase interaction and p70S6 kinase phosphorylation in a cell-context-dependent manner

We have recently shown that in colon cancer cells, Vitamin D receptor (VDR) interacts with the catalytic subunit of Ser/Thr protein phosphatases, PP1c and PP2Ac, and induces their enzymatic activity in a ligand-dependent manner. The VDR-PP1c and VDR-PP2Ac interactions were ligand independent in vivo, and 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3))-mediated increase in VDR-associated phosphatase activity resulted in dephosphorylation and inactivation of p70S6 kinase in colon cancer cells. Here, we demonstrate that in myeloid leukemia cells, 1,25(OH)(2)D(3) treatment increased the Thr389 phosphorylation of p70S6 kinase. Accordingly, 1,25(OH)(2)D(3) decreased VDR-associated Ser/Thr protein phosphatase activity by dissociating VDR-PP1c and VDR-PP2Ac interactions. Further, 1,25(OH)(2)D(3) increased the association between VDR and Thr389 phosphorylated p70S6 kinase. Finally, by using non-secosteroidal VDR ligands, we demonstrate a separation between transactivation and p70S6 kinase phosphorylation activities of VDR and show pharmacologically that p70S6 kinase phosphorylation correlates with HL-60 cell differentiation.     

Isolation of the Arabidopsis phosphoproteome using a biotin-tagging approach

Protein phosphorylation plays a key role in signal transduction in cells. Since phosphoproteins are present in low abundance, enrichment methods are required for their purification and analysis. Chemical derivatization strategies have been devised for enriching phosphoproteins and phosphopeptides. In this report, we employed a strategy that replaces the phosphate moieties on serine and threonine residues with a biotin-containing tag via a series of chemical reactions. Ribulose 1,5-bis-phosphate carboxylase/oxygenase (RUBISCO)-depleted protein extracts prepared from Arabidopsis seedlings were chemically modified for 'biotin-tagging'. The biotinylated (previously phosphorylated) proteins were then selectively isolated by avidin-biotin affinity chromatography, followed by two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). This led to the identification of 31 protein spots, representing 18 different proteins, which are implicated in a variety of cellular processes. Despite its current technical limitations, with further improvements in tools and techniques this strategy may be developed into a useful approach.     

Revealing phosphoproteins playing role in tobacco pollen activated in vitro

The transition between the quiescent mature and the metabolically active germinating pollen grain most probably involves changes in protein phosphorylation status, since phosphorylation has been implicated in the regulation of many cellular processes. Given that, only a minor proportion of cellular proteins are phosphorylated at any one time, and that phosphorylated and nonphosphorylated forms of many proteins can co‐exist within a cell, the identification of phosphoproteins requires some prior enrichment from a crude protein extract. Here, we have used metal oxide/hydroxide affinity chromatography (MOAC) based on an aluminum hydroxide matrix for this purpose, and have generated a population of phosphoprotein candidates from both mature and in vitro activated tobacco pollen grains. Both electrophoretic and nonelectrophoretic methods, allied to MS, were applied to these extracts to identify a set of 139 phosphoprotein candidates. In vitro phosphorylation was also used to validate the spectrum of phosphoprotein candidates obtained by the MOAC phosphoprotein enrichment. Since only one phosphorylation site was detected by the above approach, titanium dioxide phosphopeptide enrichment of trypsinized mature pollen crude extract was performed as well. It resulted in a detection of additional 51 phosphorylation sites giving a total of 52 identified phosphosites in this set of 139 phosphoprotein candidates.