A Rapid Screening Assay to Search for Phosphorylated Proteins in Tissue Extracts

Reversible protein phosphorylation is an essential mechanism in the regulation of diverse biological processes, nonetheless is frequently altered in disease. As most phosphoproteome studies are based on optimized in-vitro cell culture studies new methods are in need to improve de novo identification and characterization of phosphoproteins in extracts from tissues. Here, we describe a rapid and reliable method for the detection of phosphoproteins in tissue extract based on an experimental strategy that employs 1D and 2D SDS PAGE, Western immunoblotting of phosphoproteins, in-gel protease digestion and enrichment of phosphorpeptides using metal oxide affinity chromatography (MOAC). Subsequently, phosphoproteins are identified by MALDI-TOF-MS/MS with the CHCA-TL or DHB ML sample matrix preparation method and further characterized by various bioinformatic software tools to search for candidate kinases and phosphorylation-dependent binding motifs. The method was applied to mouse lung tissue extracts and resulted in an identification of 160 unique phosphoproteins. Notably, TiO₂ enrichment of pulmonary protein extracts resulted in an identification of additional 17 phosphoproteins and 20 phosphorylation sites. By use of MOAC, new phosphorylation sites were identified as evidenced for the advanced glycosylation end product-specific receptor. So far this protein was unknown to be phosphorylated in lung tissue of mice. Overall the developed methodology allowed efficient and rapid screening of phosphorylated proteins and can be employed as a general experimental strategy for an identification of phosphoproteins in tissue extracts.     

Identification of in vivo protein phosphorylation sites in human pathogen Schistosoma japonicum by a phosphoproteomic approach

Schistosome is the causative agent of human schistosomiasis and related animal disease. Reversible protein phosphorylation plays a key role in signaling processing that are vital for a cell and organism. However, it remains to be undercharacterized in schistosomes. In the present study, we characterized in vivo protein phosphorylation events in different developmental stages (schistosomula and adult worms) of Schistosoma japonicum by using microvolume immobilized metal-ion affinity chromatography (IMAC) pipette tips coupled to nanoLC-ESI-MS/MS. In total, 127 distinct phosphorylation sites were identified in 92 proteins in S. japonicum. A comparison of the phosphopeptides identified between the schistosomula and the adult worms revealed 30 phosphoproteins co-detected in both of the two worms. These proteins included several signal molecules and enzymes such as 14-3-3 protein, cysteine string protein, heat shock protein 90, epidermal growth factor receptor pathway substrate 8, proliferation-associated protein 2G4, peptidyl-prolyl isomerase G, phosphofructokinase and thymidylate kinase. Additionally, the phosphorylation sites were examined for phosphorylation specific motif and evolutionarily conservation. The study represents the first attempt to determine in vivo protein phosphorylation in S. japonicum by using a phosphoproteomic approach. The results by providing an inventory of phosphorylated proteins may facilitate to further understand the mechanisms involved in schistosome development and growth, and then may result in the development of novel vaccine candidates and drug targets for schistosomiasis control.     

Phosphoprotein analysis: from proteins to proteomes

Characterization of protein modification by phosphorylation is one of the major tasks that have to be accomplished in the post-genomic era. Phosphorylation is a key reversible modification occurring mainly on serine, threonine and tyrosine residues that can regulate enzymatic activity, subcellular localization, complex formation and degradation of proteins. The understanding of the regulatory role played by phosphorylation begins with the discovery and identification of phosphoproteins and then by determining how, where and when these phosphorylation events take place. Because phosphorylation is a dynamic process difficult to quantify, we must at first acquire an inventory of phosphoproteins and characterize their phosphorylation sites. Several experimental strategies can be used to explore the phosphorylation status of proteins from individual moieties to phosphoproteomes. In this review, we will examine and catalogue how proteomics techniques can be used to answer specific questions related to protein phosphorylation. Hence, we will discuss the different methods for enrichment of phospho-proteins and -peptides, and then the various technologies for their identification, quantitation and validation.     

The cytoplasmic phosphoproteome of the Gram-negative bacterium Campylobacter jejuni: evidence for modification by unidentified protein kinases

We have undertaken a comprehensive analysis of cytoplasmic protein phosphorylation in Campylobacter jejuni by mass spectrometric identification of phosphoproteins and localization of the sites of modification by phosphopeptide analyses. Cell extracts, enriched for phosphoproteins using Fe(III) IMAC or commercial phosphoprotein purification kits, were analyzed by 1-D and 2-D SDS-PAGE and subjected to mass fingerprinting by in-gel tryptic digestion and MALDI-TOF MS. Fifty-eight phosphopeptides were identified from 1-D gel bands by nano-LC-MS/MS and automated searching in a C. jejuni ORF database resulting in the unequivocal identification of 36 phosphoproteins of diverse function. In addition to elongation factors and chaperonins, which have been reported to be phosphorylated in other bacteria, the major phosphoproteins included bacterioferritin and superoxide dismutase. The sequences around the phosphorylated Ser and Thr residues are indicative of specific kinases being responsible for some of the modifications. However, many of the other identified proteins are enzymes that have phosphorylated substrates, including ATP, hence other modifications may arise from autophosphorylation. Comparative analyses of IMAC extracts from the Escherichia coli strain AD202 and Helicobacter pylori resulted in the identification of homologs of six of the C. jejuni phosphoproteins, though their overall phosphoproteome maps were distinctly different.     

Large-scale analysis of in vivo phosphorylated membrane proteins by immobilized metal ion affinity chromatography and mass spectrometry

Global analyses of protein phosphorylation require specific enrichment methods because of the typically low abundance of phosphoproteins. To date, immobilized metal ion affinity chromatography (IMAC) for phosphopeptides has shown great promise for large-scale studies, but has a reputation for poor specificity. We investigated the potential of IMAC in combination with capillary liquid chromatography coupled to tandem mass spectrometry for the identification of plasma membrane phosphoproteins of Arabidopsis. Without chemical modification of peptides, over 75% pure phosphopeptides were isolated from plasma membrane digests and detected and sequenced by mass spectrometry. We present a scheme for two-dimensional peptide separation using strong anion exchange chromatography prior to IMAC that both decreases the complexity of IMAC-purified phosphopeptides and yields a far greater coverage of monophosphorylated peptides. Among the identified sequences, six originated from different isoforms of the plasma membrane H(+)-ATPase and defined two previously unknown phosphorylation sites at the regulatory C terminus. The potential for large-scale identification of phosphorylation sites on plasma membrane proteins will have wide-ranging implications for research in signal transduction, cell-cell communication, and membrane transport processes.     

Quantitation of protein phosphorylation in pregnant rat uteri using stable isotope dimethyl labeling coupled with IMAC

Quantitative analysis of protein phosphorylation provides important insights into molecular signaling mechanisms and a better understanding of many cellular processes. In this study, we coupled stable isotope dimethyl labeling with immobilized metal affinity chromatography (IMAC) enrichment to quantify protein phosphorylation at MS-determined phosphorylation sites. The proposed method was first characterized using alpha- and beta-casein as two model phosphoproteins, and further applied to the analysis of pregnant rat uteri with and without treatment with 8-bromo-cGMP. Dimethyl labeling has several significant advantages: global, fast (within 5 min) and complete (near 100%). Our results indicate that the labeling has no adverse effect on the IMAC enrichment for tryptic peptides having single and multiple phosphorylation sites. Moreover, the enhanced a1 signal and the complete reaction by dimethyl labeling provide unequivocal identification of both the N-terminal amino acid and the number of the labeling site. Using these two criteria in data validation, which is particularly important for identifying phosphoproteins, we found that the confidence in interpreting dimethyl-labeled peptides had greatly increased. In the analysis of late gestation rat uteri, the abundance ratio between treated and un-treated phosphopeptide signals ranged from 0.51 to 1.69 with an average of around 1.01 +/- 0.25. The obtained ratio of the phosphorylation levels at Ser 15 of HSP27 was further confirmed by the consistent results obtained from Western blot analyses. Based on the analysis of the results, it is interesting to note that the activated cGMP dependent protein kinase G (PKG) seems to affect the phosphorylation of proteins associated with the inhibition of cell migration and proliferation, redistribution of actin-associated proteins, and the increase of protein synthesis in late-gestation uteri. These observations provide important evidence suggesting that activated PKG may play a critical role in the shift of pregnant uteri from proliferative to hypertrophic states.     

Precipitation by lanthanum ions: a straightforward approach to isolating phosphoproteins

Posttranslational modification (PTM) of proteins, particularly phosphorylation, is a key element in the regulation of cell functions. In many signal transduction processes, PTM is a pivotal step. Various analytical methods have been proposed for the identification of phosphoproteins; however, most of these methods require sophisticated equipment. Here we present an easily applicable method of phosphoprotein enrichment. This method is based on single-step precipitation by lanthanum chloride and allows subsequent protein identification by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-TOF-MS). The method proved its suitability for the isolation of phosphoproteins from frozen tissue and cultured cells samples after cell lysis in various buffer systems (urea/thiourea and EGTA/EDTA). The tests revealed that the isolation of phosphoproteins can be achieved with high efficiency even from complex protein mixtures. Our results indicate that lanthanum-based enrichment of phosphoproteins can be a useful tool in phosphoproteomic studies.     

Reference-facilitated phosphoproteomics: fast and reliable phosphopeptide validation by microLC-ESI-Q-TOF MS/MS

Recent advances in instrument control and enrichment procedures have enabled us to quantify large numbers of phosphoproteins and record site-specific phosphorylation events. An intriguing problem that has arisen with these advances is to accurately validate where phosphorylation events occur, if possible, in an automated manner. The problem is difficult because MS/MS spectra of phosphopeptides are generally more complicated than those of unmodified peptides. For large scale studies, the problem is even more evident because phosphorylation sites are based on single peptide identifications in contrast to protein identifications where at least two peptides from the same protein are required for identification. To address this problem we have developed an integrated strategy that increases the reliability and ease for phosphopeptide validation. We have developed an off-line titanium dioxide (TiO(2)) selective phosphopeptide enrichment procedure for crude cell lysates. Following enrichment, half of the phosphopeptide fractionated sample is enzymatically dephosphorylated, after which both samples are subjected to LC-MS/MS. From the resulting MS/MS analyses, the dephosphorylated peptide is used as a reference spectrum against the original phosphopeptide spectrum, in effect generating two peptide spectra for the same amino acid sequence, thereby enhancing the probability of a correct identification. The integrated procedure is summarized as follows: 1) enrichment for phosphopeptides by TiO(2) chromatography, 2) dephosphorylation of half the sample, 3) LC-MS/MS-based analysis of phosphopeptides and corresponding dephosphorylated peptides, 4) comparison of peptide elution profiles before and after dephosphorylation to confirm phosphorylation, and 5) comparison of MS/MS spectra before and after dephosphorylation to validate the phosphopeptide and its phosphorylation site. This phosphopeptide identification represents a major improvement as compared with identifications based only on single MS/MS spectra and probability-based database searches. We investigated an applicability of this method to crude cell lysates and demonstrate its application on the large scale analysis of phosphorylation sites in differentiating mouse myoblast cells.     

Quantitative phosphoproteomic profiling of fiber differentiation and initiation in a fiberless mutant of cotton

Background

The cotton (Gossypium spp.) fiber cell is an important unicellular model for studying cell differentiation. There is evidence suggesting that phosphorylation is a critical post-translational modification involved in regulation of a wide range of cell activities. Nevertheless, the sites of phosphorylation in G. hirsutum and their regulatory roles in fiber cell initiation are largely unknown. In this study, we employed a mass spectrometry-based phosphoproteomics to conduct a global and site-specific phosphoproteome profiling between ovules of a fuzzless-lintless (fl) Upland cotton (G. hirsutum) mutant and its isogenic parental wild type (WT) at -3 and 0 days post-anthesis (DPA).

Results

A total of 830 phosphopeptides and 1,592 phosphorylation sites from 619 phosphoproteins were identified by iTRAQ (isobaric tags for relative and absolute quantitation). Of these, 76 phosphoproteins and 1,100 phosphorylation sites were identified for the first time after searching the P³DB public database using the BLAST program. Among the detected phosphopeptides, 69 were differentially expressed between the fl mutant and its WT in ovules at -3 and 0 DPA. An analysis using the Motif-X program uncovered 19 phosphorylation motifs, 8 of which were unique to cotton. A further metabolic pathway analysis revealed that the differentially phosphorylated proteins were involved in signal transduction, protein modification, carbohydrate metabolic processes, and cell cycle and cell proliferation.

Conclusions

Our phosphoproteomics-based research provides the first global overview of phosphorylation during cotton fiber initiation, and also offers a helpful dataset for elucidation of signaling networks in fiber development of G. hirsutum.

Electronic supplementary material

The online version of this article (doi: 10.1186/1471-2164-15-466) contains supplementary material, which is available to authorized users.     

Drosophila CK2 regulates eye morphogenesis via phosphorylation of E(spl)M8

The Notch effector E(spl)M8 is phosphorylated at Ser159 by CK2, a highly conserved Ser/Thr protein kinase. We have used the Gal4-UAS system to assess the role of M8 phosphorylation during bristle and eye morphogenesis by employing a non-phosphorylatable variant (M8SA) or one predicted to mimic the 'constitutively' phosphorylated protein (M8SD). We find that phosphorylation of M8 does not appear to be critical during bristle morphogenesis. In contrast, only M8SD elicits a severe 'reduced eye' phenotype when it is expressed in the morphogenetic furrow of the eye disc. M8SD elicits neural hypoplasia in eye discs, elicits loss of phase-shifted Atonal-positive cells, i.e. the 'founding' R8 photoreceptors, and consequently leads to apoptosis. The ommatidial phenotype of M8SD is similar to that in Nspl/Y; E(spl)D/+ flies. E(spl)D, an allele of m8, encodes a truncated protein known as M8*, which, unlike wild type M8, displays exacerbated antagonism of Atonal via direct protein-protein interactions. In line with this, we find that the M8SD-Atonal interaction appears indistinguishable from that of M8*-Atonal, whereas interaction of M8 or M8SA appears marginal, at best. These results raise the possibility that phosphorylation of M8 (at Ser159) might be required for its ability to mediate 'lateral inhibition' within proneural clusters in the developing retina. This is the first identification of a dominant allele encoding a phosphorylation-site variant of an E(spl) protein. Our studies uncover a novel functional domain that is conserved amongst a subset of E(spl)/Hes repressors in Drosophila and mammals, and suggests a potential role for CK2 during retinal patterning.     

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.     

Large-scale phosphorylation analysis of alpha-factor-arrested Saccharomyces cerevisiae

Protein phosphorylation is essential for numerous cellular processes. Large-scale profiling of phosphoproteins continues to enhance the depth and speed at which we understand these processes. The development of effective phosphoprotein and peptide enrichment techniques and improvements to mass spectrometric instrumentation have intensified phosphoproteomic research in recent years, leading to unprecedented achievements. Here, we describe a large-scale phosphorylation analysis of alpha-factor-arrested yeast. Using a multidimensional separation strategy involving preparative SDS-PAGE for prefractionation, in-gel digestion with trypsin, and immobilized metal affinity chromatography (IMAC) enrichment of phosphopeptides, followed by LC-MS/MS analysis employing a hybrid LTQ-Orbitrap mass spectrometer, we were able to catalog a substantial portion of the phosphoproteins present in yeast whole-cell lysate. This analysis yielded the confident identification of 2288 nonredundant phosphorylation sites from 985 proteins. The ambiguity score (Ascore) algorithm was utilized to determine the certainty of site localization for the entire data set. In addition, the size of the data set permitted extraction of known and novel kinase motifs using the Motif-X algorithm. Finally, a large number of members of the pheromone signaling pathway were found as phosphoproteins and are discussed.     

Phosphoproteomic analysis of the mouse brain cytosol reveals a predominance of protein phosphorylation in regions of intrinsic sequence disorder

We analyzed the mouse forebrain cytosolic phosphoproteome using sequential (protein and peptide) IMAC purifications, enzymatic dephosphorylation, and targeted tandem mass spectrometry analysis strategies. In total, using complementary phosphoenrichment and LC-MS/MS strategies, 512 phosphorylation sites on 540 non-redundant phosphopeptides from 162 cytosolic phosphoproteins were characterized. Analysis of protein domains and amino acid sequence composition of this data set of cytosolic phosphoproteins revealed that it is significantly enriched in intrinsic sequence disorder, and this enrichment is associated with both cellular location and phosphorylation status. The majority of phosphorylation sites found by MS were located outside of structural protein domains (97%) but were mostly located in regions of intrinsic sequence disorder (86%). 368 phosphorylation sites were located in long regions of disorder (over 40 amino acids long), and 94% of proteins contained at least one such long region of disorder. In addition, we found that 58 phosphorylation sites in this data set occur in 14-3-3 binding consensus motifs, linear motifs that are associated with unstructured regions in proteins. These results demonstrate that in this data set protein phosphorylation is significantly depleted in protein domains and significantly enriched in disordered protein sequences and that enrichment of intrinsic sequence disorder may be a common feature of phosphoproteomes. This supports the hypothesis that disordered regions in proteins allow kinases, phosphatases, and phosphorylation-dependent binding proteins to gain access to target sequences to regulate local protein conformation and activity.     

Mapping sites of protein phosphorylation by mass spectrometry utilizing a chemical-enzymatic approach: characterization of products from alpha-S1 casein phosphopeptides

A novel chemical-enzymatic approach was developed to facilitate identification of phosphorylation sites in isolated phosphoproteins. ESI-TOF mass spectrometry was used to characterize products from the chemical-enzymatic cleavage of specific phosphorylation sites in bovine alpha-S1 casein and synthetic phosphopeptides containing substitutions at a single phosphorylation site. Further refinements to this approach for identification of protein phosphorylation sites and its utility for the quantification of phosphopeptides by isotope-dilution mass spectrometry are presented.     

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.     

Analysis of protein phosphorylation by mass spectrometry

The reversible phosphorylation of proteins is recognized as an essential post-translational modification regulating cell signaling and ultimately function of biological systems. Detection of phosphopeptides and localization of phosphorylation sites remains quite a challenge, even if the protein is purified to near homogeneity. Mass spectrometry has become a vital technique that is routinely utilized for the identification of proteins from whole cell lysates. Nonetheless, due to the minimal amount of phosphorylation found on proteins, enrichment steps for isolating phosphopeptides from complex mixtures have been the focus of many research groups world-wide. In this review, we describe some current methods for the enrichment of phosphopeptides that are compatible with mass spectrometry for assignment of phosphorylation sites. Phosphorylation modifications on proteins and peptides are either directly isolated by solid-phase approaches or chemically modified for selective isolation and/or improved characterization by mass spectrometry. These strategies hold the potential for rapid and sensitive profiling of phosphoproteins from a variety of sources and cellular conditions.     

A rapid method for capture and identification of immunogenic proteins in Bordetella pertussis enriched membranes fractions: a fast-track strategy applicable to other microorganisms

Mass spectrometry (MS) coupled with 1-D and 2-D electrophoresis can be utilized to detect and identify immunogenic proteins, but these methods are laborious and time-consuming. We describe an alternative, simple, rapid gel-free strategy to identify multiple immunogenic proteins from Bordetella pertussis (Bp). It couples immunoprecipitation to nano liquid chromatography- tandem mass spectrometry (IP-nLC-MS/MS) and is significantly both time- and labor-saving. We developed a gel-free magnetic bead-based immunoprecipitation (IP) method using different NP-40/PBS concentrations in which solubilized proteins of Bp Tohama I membrane fractions were precipitated with polyclonal rabbit anti-Bp whole cell immune sera. Immune complexes were analyzed by MS and Scaffold analysis (> 95% protein identification probability). Total immunoproteins identified were 50, 63 and 49 for 0.90%, 0.45% and 0.22% NP-40/PBS buffer concentrations respectively. Known Bp proteins identified included pertactin, serotype 2 fimbrial subunit and filamentous hemagglutinin. As proof of concept that this gel-free protein immunoprecipitation method enabled the capture of multiple immunogenic proteins, IP samples were also analyzed by SDS-PAGE and immunoblotting. Bypassing gels and subjecting immunoprecipitated proteins directly to MS is a simple and rapid antigen identification method with relatively high throughput. IP-nLC-MS/MS provides a novel alternative approach for current methods used for the identification of immunogenic proteins.     

Label-free liquid chromatography–tandem mass spectrometry analysis with automated phosphopeptide enrichment reveals dynamic human milk protein phosphorylation during lactation

Protein phosphorylation is a critical posttranslational modification that affects cell–cell signaling and protein function. However, quantifying the relative site-specific changes of phosphorylation occupancies remains a major issue. An online enrichment of phosphopeptides using titanium dioxide incorporated in a microchip liquid chromatography device was used to analyze trypsin-digested human milk proteins with mass spectrometry. The method was validated with standards and used to determine the dynamic behavior of protein phosphorylation in human milk from the first month of lactation. α-Casein, β-casein, osteopontin, and chordin-like protein 2 phosphoproteins were shown to vary during this lactation time in an independent manner. In addition, changes in specific regions of these phosphoproteins were found to vary independently. Novel phosphorylation sites were discovered for chordin-like protein 2, α-lactalbumin, β-1,4-galactosyl transferase, and poly-Ig (immunoglobulin) receptor. Coefficients of variation for the quantitation were comparable to those in other contemporary approaches using isotopically labeled peptides, with a median value of 11% for all phosphopeptide occupancies quantified.