Phosphoprotein Purification Research Articles

Microfluidic immobilized metal affinity chromatography based on Ti(IV)-decorated silica microspheres for purification of phosphoproteins

A silica-based immobilized metal affinity chromatography (IMAC) sorbent with the morphological properties suitable for purification of large phosphorylated biomolecules was synthesized. The sorbent was designed in the form of monodisperse-porous silica microspheres, 5.3 μm in size, having bimodal pore size distribution with a large median pore size (40 nm) and high surface area (163 m2/g) decorated with Ti(IV) cations (i.e. Ti(IV)@THSPMP@SiO2 microspheres). The decoration of silica microspheres with Ti(IV) cations was made by using 3-(trihydroxysilyl)propyl methylphosphonate (THSPMP) as a bifunctiontional linker, by preserving their bimodal pore size distribution. The mesopores provided a large surface area for parking of adsorbed phosphoproteins as large phosphorylated biomolecules while the intraparticular transport of phosphoproteins was facilitated by the macropores providing a large median pore size. High equilibrium adsorption capacity and high desorption yield in the purification of phosphoproteins were obtained using Ti(IV)@THSPMP@SiO2 microspheres as the sorbent in batch- and microfluidic-IMAC systems. The phosphoproteins, α-casein and β-casein were isolated from milk and human serum with almost quantitative yields and high purity in the batch IMAC system. The appropriate microcolumn permeability (3.66 × 10−14 m2) originating from its appropriate average diameter (5.3 μm), high porosity (0.948 cm3/g) and high surface area (163 m2/g) of Ti(IV)@THSPMP@SiO2 microspheres makes the synthesized sorbent a promising stationary phase for dynamic chromatography. Hence, a new phosphoprotein enrichment format, a microfluidic IMAC system was constructed and successfully operated for highly selective purification of phosphoproteins from non-fat milk as a complex sample. The microfluidic-IMAC system is a promising tool particularly for phosphoproteomic applications performed using samples in microliter or nanoliter scale, also involving an on-line connection of purification unit to LC-MS for the identification of large phosphorylated biomolecules enriched.

Exploring protein phosphorylation by combining computational approaches and biochemical methods

Post-translational modifications of proteins expand their functional diversity, regulating the response of cells to a variety of stimuli. Among these modifications, phosphorylation is the most ubiquitous and plays a prominent role in cell signaling. The addition of a phosphate often affects the function of a protein by altering its structure and dynamics. However, these alterations are often difficult to study and the functional and structural implications remain unresolved. New approaches are emerging to overcome common obstacles related to the production and manipulation of these samples. Here, we summarize the available methods for phosphoprotein purification and phosphomimetic engineering, highlighting the advantages and disadvantages of each. We propose a general workflow for protein phosphorylation analysis combining computational and biochemical approaches, building on recent advances that enable user-friendly and easy-to-access Molecular Dynamics simulations. We hope this innovative workflow will inform the best experimental approach to explore such post-translational modifications. We have applied this workflow to two different human protein models: the hemeprotein cytochrome c and the RNA binding protein HuR. Our results illustrate the usefulness of Molecular Dynamics as a decision-making tool to design the most appropriate phosphomimetic variant.

Scalable, Non-denaturing Purification of Phosphoproteins Using Ga3+-IMAC: N2A and M1M2 Titin Components as Study case

The purification of phosphorylated proteins in a folded state and in large enough quantity for biochemical or biophysical analysis remains a challenging task. Here, we develop a new implementation of the method of gallium immobilized metal chromatography (Ga3+-IMAC) as to permit the selective enrichment of phosphoproteins in the milligram scale and under native conditions using automated FPLC instrumentation. We apply this method to the purification of the UN2A and M1M2 components of the muscle protein titin upon being monophosphorylated in vitro by cAMP-dependent protein kinase (PKA). We found that UN2A is phosphorylated by PKA at its C-terminus in residue S9578 and M1M2 is phosphorylated in its interdomain linker sequence at position T32607. We demonstrate that the Ga3+-IMAC method is efficient, economical and suitable for implementation in automated purification pipelines for recombinant proteins. The procedure can be applied both to the selective enrichment and to the removal of phosphoproteins from biochemical samples.

PO-522 Global proteome and phosphoprotein profiling of meningiomas reveals novel potential therapeutic targets and biomarkers

IntroductionMeningiomas are the most common primary intracranial brain tumour arising from meningeal tissue. Despite the majority of them displaying benign features, they can cause mild to severe morbidity. The current main therapeutic approach is complete tumour resection commonly with adjunct radiation therapy. However, tumour location can hamper complete resection and chemotherapies are ineffective. In this study we aim to elucidate the pathogenic signature of these tumours and identify novel molecular targets by deciphering the global proteome and phosphoprotein profile of different grades of meningiomas.Material and methodsTumour lysates were collected from grade I, II and III frozen meningioma specimens and three normal healthy human meninges. Phosphoprotein purification was performed using Qiagen® PhosphoProtein Purification Kit. Proteins were separated by SDS-PAGE followed by in-gel tryptic digestion. Extracted peptides were purified and analysed by electrospray ionisation LC-MS/MS. Raw mass spectrometry files were analysed using MaxQuantTM. Expression data were validated by Western blot and immunohistochemistry. In silico functional annotation of expression data was completed using Perseus 1.5.0.31 software suite, Ingenuity Pathway Analysis (IPA®) and DAVID 6.8.Results and discussionsWe have quantified 3888 proteins and 3074 phosphoproteins across all grades of meningioma and normal meninges. Comparative analysis identified 181 proteins and 338 phosphoproteins to be commonly significantly upregulated (log2 fold-change ≥1.5; p<0.05) among all grades vs. normal meninges, and further identified differential expression profiles between meningioma grades. Expression data was validated on samples analysed by MS and on an additional cohort of meningiomas for upregulated proteins including SET, EGFR, SRSF1, CKAP4 and HK2; and phosphoproteins including AKT1, AKT2 and RB1. Gene Ontology revealed commonly upregulated proteins to be enriched in terms including RNA helicase activity, whilst phosphoproteins were enriched in the phosphoprotein associated terms of signal complex assembly, Rho guanyl-nucleotide exchange factor activity and EGFR signalling.ConclusionIn summary, we performed a comprehensive quantitative proteomic analysis from meningioma tissue of all WHO grades compared to healthy meninges and have identified several potential candidates that may hold therapeutic potential for targeted treatment of these tumours.

Global proteome and phosphoproteome analysis of meningiomas

Meningiomas are common brain tumours arising from meningeal tissue. Despite the majority of them displaying benign features, they can cause mild to severe morbidity. The current main therapeutic approach is complete tumour resection commonly with adjunct radiation therapy. However, tumour location can hamper complete resection and chemotherapies are ineffective. In this study we aim to elucidate dysregulated pathways in meningioma pathogenesis and identify novel molecular targets by deciphering the proteome and phosphoproteome of different grades of meningiomas. Tumour lysates were collected from grade I, II and III frozen meningioma specimens and three normal healthy human meninges. Phosphoprotein purification was performed using Qiagen® PhosphoProtein Purification Kit. Proteins were separated by SDS-PAGE followed by in-gel tryptic digestion. Extracted peptides were purified and analysed by electrospray ionization LC-MS/MS. Raw mass spectrometry files were analysed using MaxQuantTM. Expression data was validated by Western blot and functionally annotated using Ingenuity® Pathway Analysis (IPA®) and DAVID 6.8. We have quantified 3888 proteins and 3074 phosphoproteins across all grades of meningioma and normal meninges. Comparative analysis confirmed 181 proteins and 338 phosphoproteins to be commonly significantly upregulated (log2 fold-change≥1.5; p<0.05) among all grades vs. normal meninges. We have successfully validated the expression data of several upregulated proteins and phosphoproteins. Gene Ontology revealed biological processes including EFGR and VEGFR signalling to be enriched in the phosphoproteome. Grade-wise comparisons identified 667 proteins and 769 phosphoproteins to be differentially expressed (p<0.05) between grade III meningiomas compared to grade II and I. We have performed a comparative proteomic analysis across all meningioma grades and identified changes in proteomic profiles between these tumours and normal healthy meninges. We will use this data to define novel targets common to all grades and specific to a grade of meningioma for future therapies.

B7-H3 combats apoptosis induced by chemotherapy by delivering signals to pancreatic cancer cells

ObjectiveThis study aimed to investigate the role of B7-H3 in chemotherapy resistance of pancreatic cancer cells and discover the potential signal transduction pathway and molecular targets involved.MethodsImmunohistochemical staining and real-time polymerase chain reaction (PCR) were used to determine the expression of B7-H3 in clinical specimens. Clinical data were applied to survival analysis. Phosphoprotein was purified from cultured Patu8988 cells using the Phosphoprotein Purification Kit. Cell apoptosis was detected using propidium iodide–Annexin V staining to investigate the relation between the expression of B7-H3 and Patu8988 cells treated with gemcitabine. Western blot was used to determine the effect of B7-H3 on the expression of proteins including extracellular signal–regulated kinase (ERK)1/2, epidermal growth factor receptor (EGFR), and Inhibitor of NF-κB(IκB) in Patu8988 cells; B7-H3 was activated by 4H7, which as an agonist monoclonal antibody to B7-H3.ResultsThe expression of B7-H3 was found to be higher in tumor tissues than in normal tissues of pancreatic carcinoma. Survival analysis revealed that patients in the low-B7-H3 expression group were likely to have a longer overall survival compared with those in the high-expression group (P < 0.05). B7-H3 activated by 4H7 could reduce gemcitabine-induced apoptosis in Patu8988 cells. Activation of B7-H3 by 4H7 induced variations in p-ERK1/2, EGFR, and IκB protein levels. When B7-H3 was upregulated, the expression levels of EGFR and p-ERK1/2 proteins significantly increased (P < 0.05), but the expression level of IκB significantly decreased (P < 0.05), especially in the gemcitabine-treated group.ConclusionThis study demonstrated that B7-H3 could deliver signals to pancreatic cancer cells to combat apoptosis induced by gemcitabine.

A Cell-Permeable ATP Analogue for Kinase-Catalyzed Biotinylation.

ATP analogues have been powerful compounds for the study of kinase-catalyzed phosphorylation. However, the cell impermeability of ATP analogues has largely limited their use to in vitro lysate-based experiments. Herein, we report the first cell-permeable ATP analogue, ATP-polyamine-biotin (APB). APB is shown to promote biotin labeling of kinase substrates in live cells and has future applications in phosphoprotein purification and analysis. More generally, these studies provide a foundation for the development of additional cell-permeable ATP analogues for cell-signaling research.

A Cell-Permeable ATP Analogue for Kinase-Catalyzed Biotinylation

ATP analogues have been powerful compounds for the study of kinase-catalyzed phosphorylation. However, the cell impermeability of ATP analogues has largely limited their use to in vitro lysate-based experiments. Herein, we report the first cell-permeable ATP analogue, ATP–polyamine–biotin (APB). APB is shown to promote biotin labeling of kinase substrates in live cells and has future applications in phosphoprotein purification and analysis. More generally, these studies provide a foundation for the development of additional cell-permeable ATP analogues for cell-signaling research.

Magnetic Affinity Microspheres with Meso-/Macroporous Shells for Selective Enrichment and Fast Separation of Phosphorylated Biomolecules

The flowerlike multifunctional affinity microspheres prepared by a facile solvothermal synthesis and subsequent calcination process consist of magnetic cores and hierarchical meso-/macroporous TiO2 shells. The hierarchical porous structure of the flowerlike affinity microspheres is constructed by the macroporous shell from the stacked mesoporous nanopetals which are assembled by small crystallites. The affinity microspheres have a relatively large specific surface area of 50.45 m(2) g(-1) and superparamagnetism with a saturation magnetization (Ms) value of 30.1 emu g(-1). We further demonstrate that they can be applied for rapid and effective purification of phosphoproteins, in virtue of their selective affinity, porous structure, and strong magnetism. In addition, the affinity microspheres can also be used for enrichment of phosphopeptides, and the selectivity is greatly improved due to the increase of mass transport and prevention of the possible "shadow effect" resulting from the smaller and deeper pores by taking advantage of the unique porous structure. Overall, this work will be highly beneficial for future applications in the isolation and identification of phosphorylated biomolecules.

Biotinylated phosphoproteins from kinase-catalyzed biotinylation are stable to phosphatases: implications for phosphoproteomics.

Kinase-catalyzed protein phosphorylation is involved in a wide variety of cellular events. Development of methods to monitor phosphorylation is critical to understand cell biology. Our lab recently discovered kinase-catalyzed biotinylation, where ATP-biotin is utilized by kinases to label phosphopeptides or phosphoproteins with a biotin tag. To exploit kinase-catalyzed biotinylation for phosphoprotein purification and identification in a cellular context, the susceptibility of the biotin tag to phosphatases was characterized. We found that the phosphorylbiotin group on peptide and protein substrates was relatively insensitive to protein phosphatases. To understand how phosphatase stability would impact phosphoproteomics research applications, kinase-catalyzed biotinylation of cell lysates was performed in the presence of kinase or phosphatase inhibitors. We found that biotinylation with ATP-biotin was sensitive to inhibitors, although with variable effects compared to ATP phosphorylation. The results suggest that kinase-catalyzed biotinylation is well suited for phosphoproteomics studies, with particular utility towards monitoring low-abundance phosphoproteins or characterizing the influence of inhibitor drugs on protein phosphorylation.

Highly efficient precipitation of phosphoproteins using trivalent europium, terbium, and erbium ions

This study describes a highly efficient method for the selective precipitation of phosphoproteins by trivalent europium, terbium, and erbium metal ions. These metal cations belong to the group of lanthanides and are known to be hard acceptors with an overwhelming preference for oxygen-containing anions such as phosphates to which they form very tight ionic bonds. The method could be successfully applied to specifically precipitate phosphoproteins from complex samples including milk and egg white by forming solid metal-protein complexes. Owing to the low solubility product of the investigated lanthanide salts, the produced metal-protein complexes showed high stability. The protein pellets were extensively washed to remove nonphosphorylated proteins and contaminants. For the analysis of proteins the pellets were first dissolved in 30 % formic acid and subjected to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS. For peptide mass-fingerprint analysis the precipitated phosphoproteins were enzymatically digested using microwave-assisted digestion. The method was found to be highly specific for the isolation and purification of phosphoproteins. Protein quantification was performed by colorimetric detection of total precipitated phosphoproteins and revealed more than 95 % protein recovery for each lanthanide salt.

Phosphoproteomic Analysis of Mouse Thymoma Cells Treated With Tributyltin Oxide: TBTO Affects Proliferation and Energy Sensing Pathways

We report the results of phosphoproteomic analysis of mouse thymoma cells treated with tributyltin oxide (TBTO), an immunotoxic compound. After cell lysis, phosphoproteins were isolated using Phosphoprotein Purification Kit, separated by SDS-PAGE and subsequently digested with trypsin. Phosphopeptides were enriched employing titanium dioxide, and the obtained fractions were analyzed by nano-LC-MS/MS. A total of 160 phosphoproteins and 328 phosphorylation sites were identified in thymoma cells. Among the differentially phosphorylated proteins identified in TBTO-treated cells were key enzymes, which catalyze rate-limiting steps in pathways that are sensitive to cellular energy status. These proteins included acetyl-CoA carboxylase isoform 1, which catalyzes the rate-limiting step of fatty acid synthesis. Another enzyme was glutamine: fructose-6-phosphate amidotransferase, GFAT1, the first and rate-limiting enzyme for the hexoamine synthesis pathway. Pyruvate dehydrogenase (PDH), a multicomplex enzyme that catalyzes the rate-limiting step of aerobic oxidation of fuel carbohydrates, was identified in both TBTO-treated and control cells; however, phosphorylation at residue S293, known to inhibit PDH activity, was identified only in control cells. A lower expression level of ribosomal protein S6 kinase 1, a downstream kinase of the mammalian target of rapamycin signaling pathway implicated in protein synthesis through phosphorylation of 40 ribosomal S6, was observed in the treated cells. Giant kinases like AMP-activated protein kinase (AMPK) and cAMP-dependent protein kinase (PKAR1A), which are known to mediate the phosphorylation of these enzymes, were identified in TBTO-treated cells. Downregulation of proteins, such as MAPK, matrin-3 and ribonucleotide reductase, subunit RRM2, which are implicated in cell proliferation, was also observed in TBTO-treated cells. Together, the results show that TBTO affects proliferation and energy sensor pathways.

Mass Spectrometry-Based Analysis of Proteomic Changes in the Root Tips of Flooded Soybean Seedlings

Flooding injury is a major problem in soybean cultivation. A proteomics approach was used to clarify the occurrence of changes in protein expression level and phosphorylation in soybeans under flooding stress. Two-day-old seedlings were flooded for 1 day, proteins were extracted from root tips of the seedlings and digested with trypsin, and their expression levels and phosphorylation states were compared to those of untreated controls using mass spectrometry-based proteomics techniques. Phosphoproteins were enriched using a phosphoprotein purification column prior to digestion and mass spectrometry. The expression of proteins involved in energy production increased as a result of flooding, while expression of proteins involved in protein folding and cell structure maintenance decreased. Flooding induced changes of phosphorylation status of proteins involved in energy generation, protein synthesis and cell structure maintenance. The response to flooding stress may be regulated by both modulation of protein expression and phosphorylation state. Energy-demanding and production-related metabolic pathways may be particularly subject to regulation by changes in protein phosphorylation during flooding.

Effects Of Leucine Supplementation In The Protein Synthesis Signalling Pathways Of Soleus And Edl Muscle In Young And Old Rats

PURPOSE: The objective of this study was to carry out a comparative phosphoproteome analysis of soleus and EDL muscles of young (6 months) and old rats (22 months) supplemented with low dose and high dose of leucine. METHODS: Food-deprived (18h) rats (both groups) were assigned randomly to one of three treatments: control (saline), low dose leucine (0.135g/kg) or high dose of leucine (1.35g/kg) by oral gavage. Thirty min after the administration of the amino acid, the rats were killed by decapitation and EDL and soleus muscles were collected and stored at -80°C. The proteins were extracted in lysis buffer using a cell disruptor and phosphorylated proteins were separated using a phosphoprotein purification kit. Differential analysis was carried out using SDS-PAGE and the ImageJ software. The proteins were excised and in-gel digested with trypsin. Mass spectrometry was performed using a Capillary LC-Q-TOF - MS/MS. Mascot Daemon was used to process spectra and identify proteins using the IPIrat 2010 database. An identification was considered as valid based on the MASCOT algorithm threshold given for each protein search (p < 0.05). RESULTS: Eighteen protein spots were identified and 9 of these were differentially expressed. Calsequestrin 1, ATP synthase subunit alfpha, alpha-crystallin B chain, myosin regulatory light chain 2, troponin and hemoglobin subunit beta -1 were induced in muscles of old rats given high doses of leucine, while acta 1 actin, troponin I and carbonic anhydrase 3 were induced in two conditions: young muscles given low doses of leucine and old muscles given high doses of leucine. CONCLUSION: The phosphorylation profile of young muscles given low doses of leucine was partially similar to the profile of old muscles given high doses of leucine. On the other hand, there was no detectable change in the phosphorylation profile of young muscles given high doses or old muscles given low doses of leucine when compared to the controls, indicating that the response may be dose-dependent. The role of leucine in inducing the phosphorylation of these proteins is still unknown and is currently under investigation. Furthermore, western-blot and histochemistry analysis are being performed. Supported by FAPESP: 2009/52022-2 and Laboratório Nacional de Luz Síncrotron.

Phosphoprotein affinity purification identifies proteins involved in S-adenosyl-L-methionine-induced enhancement of antibiotic production in Streptomyces coelicolor

Streptomycetes are the major natural source of clinical antibiotics. The enhanced secondary metabolite production of many streptomycetes by S-adenosylmethionine (SAM) in previous studies suggested the existence of a common SAM regulatory effect. We screened nine proteins using the phosphoprotein purification column from Streptomyces coelicolor. Among them, genes (SCO5477, SCO5113, SCO4647, SCO4885 and SCO1793) for five proteins were disrupted by insertion mutation. The undecylprodigiosin and actinorhodin productions were changed in all mutations. The SAM-induced enhancement of actinorhodin production was abolished by all mutations except SCO4885 mutation, which reduced the production of actinorhodin and undecylprodigiosin with SAM treatment. This study demonstrates that phosphoprotein affinity purification can be used as a screening method to identify the proteins involved SAM signaling.

PpsA, TufA, and Glutaminase Were Modulated under Yop Secretion Conditions in Yersinia enterocolitica

Yersinia enterocolitica expresses the Ysc type III secretion system (TTSS) for delivery of effector proteins (Yops) to host cells to manipulate immune system of the host. The study on the physiological change of Y. Enterocolitica is important in determining how cells efficiently support the expression of virulence factors and adapt to the inside of hosts, which is completely different from the outside environment. Ysc TTSS also secretes Yops into medium when cells are grown in calcium-deficient medium at 37°C. In the present work, a phosphoprotein purification kit was used to selectively enrich proteins whose characteristics changed under the Yop production conditions. Phosphoenol pyruvate synthase (PpsA), elongation factor Tu (TufA), and glutaminase were identified by matrix-assisted laser desorption/ionization time-of-flight. These results showed that the affinity to column and/or the steady-state level of PpsA, TufA, and glutaminase are altered under the Yop secretion conditions and suggested that any protein characteristic of these three proteins was changed under the conditions for the virulence factor expression suggested that certain characteristics of these three proteins were changed. This work demonstrated that specific protein purification method can be used to identify proteins from a complex protein mixture of cell extracts with emphasis on the specific characteristics of proteins.

HER2 Carboxyl-terminal Fragments Regulate Cell Migration and Cortactin Phosphorylation

A group of breast cancer patients with a higher probability of developing metastasis expresses a series of carboxyl-terminal fragments (CTFs) of the tyrosine kinase receptor HER2. One of these fragments, 611-CTF, is a hyperactive form of HER2 that constitutively establishes homodimers maintained by disulfide bonds, making it an excellent model to study overactivation of HER2 during tumor progression and metastasis. Here we show that expression of 611-CTF increases cell motility in a variety of assays. Since cell motility is frequently regulated by phosphorylation/dephosphorylation, we looked for phosphoproteins mediating the effect of 611-CTF using two alternative proteomic approaches, stable isotope labeling with amino acids in cell culture and difference gel electrophoresis, and found that the latter is particularly well suited to detect changes in multiphosphorylated proteins. The difference gel electrophoresis screening identified cortactin, a cytoskeleton-binding protein involved in the regulation of cell migration, as a phosphoprotein probably regulated by 611-CTF. This result was validated by characterizing cortactin in cells expressing this HER2 fragment. Finally, we showed that the knockdown of cortactin impairs 611-CTF-induced cell migration. These results suggest that cortactin is a target of 611-CTF involved in the regulation of cell migration and, thus, in the metastatic behavior of breast tumors expressing this CTF.

Identification of protein kinase substrates by proteomic approaches

This review describes the current status of proteomic approaches to identify kinase substrates, which may lead to valuable medical applications. It guides the reader towards various methods using 2DE and liquid chromatography-tandem mass spectrometry. Dynamic changes of phosphorylation during extracellular stimuli can be quantitatively monitored by both technologies. Among appropriate prefractionation procedures, the purification of phosphoproteins and phosphopeptides is an absolute step for success. The temporal change and stoichiometry of phosphorylation are the important criteria to evaluate the physiological meaning of the reaction. Kinase substrates can also be identified by in vitro phosphorylation systems employing protein arrays, fractionated lysates, genetically engineered kinases and phage libraries. The final section contains an expert opinion on the current strategies and the issues we are going to challenge in the next 5 years.

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.

Purification of phosphoproteins by immobilized metal affinity chromatography and its application to phosphoproteome analysis

Prefractionation procedures facilitate the identification of lower-abundance proteins in proteome analysis. Here we have optimized the conditions for immobilized metal affinity chromatography (IMAC) to enrich for phosphoproteins. The metal ions, Ga(III), Fe(III), Zn(II), and Al(III), were compared for their abilities to trap phosphoproteins; Ga(III) was the best. Detailed analyses of the pH and ionic strength for IMAC enabled us to determine the optimal conditions (pH 5.5 and 0.5 m NaCl). When whole cell lysates were fractionated in this way, about one-tenth of the total protein was recovered in the eluate, and the recovery of phosphorylated extracellular signal-regulated kinase (ERK) was more than 90%. Phosphorylated forms of ribosomal S6 kinase (RSK) and Akt were also enriched efficiently under the same conditions. Our Ga(III) IMAC and a commercially available purification kit for phosphoproteins performed similarly, with a slight difference in the spectrum of phosphoproteins. When phosphoproteins enriched from NIH3T3 cells in which ERK was either activated or suppressed were analyzed by two-dimensional fluorescence difference gel electrophoresis, phosphorylated ERK was detected as discrete spots unique to ERK-activated cells, which overlapped with surrounding spots in the absence of prefractionation. We applied the same technique to search for Akt substrates and identified Abelson interactor 1 as a novel potential target. These results demonstrate the efficacy of phosphoprotein enrichment by IMAC and suggest that this procedure will be of general use in phosphoproteome research.