Cerium ion-chelated magnetic silica microspheres for enrichment and direct determination of phosphopeptides by matrix-assisted laser desorption ionization mass spectrometry

In this study, we employed, for the first time, the Ce4+-chelated magnetic silica microspheres to selectively concentrate phosphopeptides from protein digest products. Cerium ions were chelated onto magnetic silica microspheres using the strategy we established before. After enrichment, the phosphopeptide-conjugated magnetic microspheres were separated from the sample solution just by using a magnet. With the optimized enrichment conditions, the performance of the Ce4+-chelated magnetic microspheres was compared with the Fe3+-chelated microspheres using tryptic digested peptides originating from ovalbumin, a five protein mixture containing phosphoproteins and nonphosphoproteins, as well as a mixture of beta-casein and BSA with a molar ratio of 1:50. Compared to Fe3+, Ce4+-chelated magnetic microspheres exhibited more selective isolation ability for concentrating phosphopeptides from complex mixtures. Even when the amount of the tryptic digest product of BSA is 50 times higher than that of beta-casein in the sample solution, the trace phosphopeptides derived from beta-casein can still be concentrated effectively by the Ce4+-chelated magnetic microspheres in only 30 s. Furthermore, we initially utilized the Ce4+-chelated magnetic microspheres to directly enrich phosphopeptides from human serum without extra purification steps or tedious treatment, which opens up a possibility for their further application in phosphoproteomics.     

Large-scale phosphoproteome analysis of human liver tissue by enrichment and fractionation of phosphopeptides with strong anion exchange chromatography

The mixture of phosphopeptides enriched from proteome samples are very complex. To reduce the complexity it is necessary to fractionate the phosphopeptides. However, conventional enrichment methods typically only enrich phosphopeptides but not fractionate phosphopeptides. In this study, the application of strong anion exchange (SAX) chromatography for enrichment and fractionation of phosphopeptides was presented. It was found that phosphopeptides were highly enriched by SAX and majority of unmodified peptides did not bind onto SAX. Compared with Fe(3+) immobilized metal affinity chromatography (Fe(3+)-IMAC), almost double phosphopeptides were identified from the same sample when only one fraction was generated by SAX. SAX and Fe(3+)-IMAC showed the complementarity in enrichment and identification of phosphopeptides. It was also demonstrated that SAX have the ability to fractionate phosphopeptides under gradient elution based on their different interaction with SAX adsorbent. SAX was further applied to enrich and fractionate phosphopeptides in tryptic digest of proteins extracted from human liver tissue adjacent to tumorous region for phosphoproteome profiling. This resulted in the highly confident identification of 274 phosphorylation sites from 305 unique phosphopeptides corresponding to 168 proteins at false discovery rate (FDR) of 0.96%.     

Rapid enrichment of phosphopeptides from tryptic digests of proteins using iron oxide nanocomposites of magnetic particles coated with zirconia as the concentrating probes

Iron oxide nanocomposites of magnetic particles coated with zirconia were used as affinity probes to selectively concentrate phosphopeptides from tryptic digests of alpha- and beta-caseins, milk, and egg white to exemplify the enrichment of phosphopeptides from complex samples. Phosphopeptides, in quantities sufficient for characterization by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS), were enriched by the affinity probes within only 30 s. The affinity probe-target species conjugates were separated from the sample solution simply by applying an external magnetic field. The detection limit for tryptic digest of beta-casein using this approach is approximately 45 fmol. Furthermore, we combined this enrichment method with a rapid enzymatic digestion method, that is, microwave-assisted enzymatic digestion using magnetic particles as the microwave absorbers, to speed up the tryptic digest reactions. Thus, we alternatively enriched phosphoproteins on the zirconia-coated particles followed by mixing with trypsin and heated the mixture in a microwave oven for 1 min. The particles remaining in the mixture were used as affinity probes to selectively enrich phosphopeptides from the tryptic digestion product by pipetting, followed by characterization using MALDI MS. Using the bifunctional zirconia-coated magnetic particles as both the affinity probes and the microwave absorbers could greatly reduce the time for the purification and characterization of phosphopeptides from complex samples.     

Integration of an on-line protein digestion microreactor to a nanoelectrospray emitter for peptide mapping

A method for integrating nanoelectrospray mass spectrometry with a microreactor for on-line digestion and fast peptide mass mapping from dilute protein samples is presented. Fused silica capillaries (i.d. 50 microm, o.d. 360 microm) are employed as the digestion microreactor and the nanoelectrospray emitter by immobilizing trypsin onto the surface of the inner wall of the fused silica capillary tubing. The procedure is demonstrated using solutions of 1pmol/mul angiotensin II, cytochrome c, hemoglobin, and beta-casein. Because the inner walls of the capillaries are modified by covalent chemical bonds, the adsorption of peptides and proteins to the inner walls of the capillaries is suppressed. This procedure was performed with solutions as dilute as 1fmol/mul (1nM) cytochrome c. This method shows generation of tryptic peptides with sequence coverage up to 90% within minutes; trypsin autolysis products are not detected. In addition, the immobilized enzyme can be cleaned easily, enabling the microreactor to be reused for nanoelectrospray.     

Preparation of Fe3O4@ZrO2 core-shell microspheres as affinity probes for selective enrichment and direct determination of phosphopeptides using matrix-assisted laser desorption ionization mass spectrometry

Fe3O4@ZrO2 microspheres with well-defined core-shell structure were prepared and applied for the highly selective enrichment of phosphopeptides from tryptic digest product of proteins. To successfully coat iron oxide microspheres with uniform zirconia shell, magnetic Fe3O4 microspheres were first synthesized via a solvothermal reaction, followed by being coated with a thin layer of carbon by polymerization and carbonization of glucose through hydrothermal reaction. Finally, with the use of the Fe3O4@C microspheres as templates, zirconium isopropoxide was prehydrolyzed and absorbed onto the microspheres and eventually converted into zirconia by calcinations. The as-prepared Fe3O4@ZrO2 core-shell microspheres were used as affinity probes to selectively concentrate phosphopeptides from tryptic digest of beta-casein, casein, and five protein mixtures to exemplify their selective enrichment ability of phosphopeptides from complex protein samples. In only 0.5 min, phosphopeptides sufficient for characterization by MALDI-MS could be enriched by the Fe3O4@ZrO2 microspheres. The results demonstrate that Fe3O4@ZrO2 microspheres have the excellent selective enrichment capacity for phosphopeptides from complex samples. The performance of the Fe3O4@ZrO2 microspheres was further compared with commercial IMAC beads for the enrichment of peptides originating from tryptic digestion of beta-casein and bovine serum albumin (BSA) with a molar ratio of 1:50, and the results proved a stronger selective ability of Fe3O4@ZrO2 microspheres over IMAC beads. Finally, the Fe3O4@ZrO2 microspheres were successfully utilized for enrichment of phosphopeptides from human blood serum without any other purification procedures.     

Fe3+ immobilized metal affinity chromatography with silica monolithic capillary column for phosphoproteome analysis

Immobilized metal affinity chromatography (IMAC) is a commonly used technique for phosphoproteome analysis due to its high affinity for adsorption of phosphopeptides. Miniaturization of IMAC column is essential for the analysis of a small amount of sample. Nanoscale IMAC column was prepared by chemical modification of silica monolith with iminodiacetic acid (IDA) followed by the immobilization of Fe3+ ion inside the capillary. It was demonstrated that Fe3+-IDA silica monolithic IMAC capillary column could specifically capture the phosphopeptides from tryptic digest of alpha-casein with analysis by MALDI-TOF MS. The silica monolithic IMAC capillary column was manually coupled with nanoflow RPLC/nanospray ESI mass spectrometer (muRPLC-nanoESI MS) for phosphoproteome analysis. The system was validated by analysis of standard phosphoproteins and then it was applied to the analysis of protein phosphorylation in mouse liver lysate. Besides MS/MS spectra, MS/MS/MS spectra were also collected for neutral loss peak. After database search and manual validation with conservative criteria, 29 singly phosphorylated peptides were identified by analyzing a tryptic digest of only 12 mug mouse liver lysate. The results demonstrated that the silica monolithic IMAC capillary column coupled with muRPLC-nanoESI MS was very suitable for the phosphoproteome analysis of minute sample.     

Designed synthesis of fluorous‐functionalized magnetic mesoporous microspheres for specific enrichment of phosphopeptides with fluorous derivatization

In this work, for the first time, perfluorinated magnetic mesoporous microspheres were designed and synthesized for the highly specific enrichment of fluorous‐derivatized phosphopeptides through the unique fluorine–fluorine interactions. The perfluorinated magnetic mesoporous microspheres were prepared through a surfactant‐mediated one‐pot approach and successfully applied to the selective extraction of fluorous‐derivatized phosphopeptides from β‐casein tryptic digest, protein mixtures, and human serum. Thanks to the hydrophilic silanol groups exposed on the surface, perfluorinated groups modified in the pore channels and the magnetic cores, the flourous‐functionalized magnetic microspheres exhibited excellent dispersibility, specificity toward fluorous‐derivatized phosphopeptides while facilitated separation procedures. The novel composites achieved a high selectivity of 1:1000 toward nonphosphorylated peptides and proved to be practicable in the enrichment of endogenous phosphopeptides in the human serum sample.     

Rapid enrichment of phosphopeptides and phosphoproteins from complex samples using magnetic particles coated with alumina as the concentrating probes for MALDI MS analysis

In this study, we used nanocomposite magnetic particles coated with alumina as the affinity probes to selectively concentrate phosphorylated peptides and proteins from a low volume of sample solution. Tryptic digest products of phosphoproteins including alpha and beta-caseins, human protein phosphatase inhibitor 1, nonfat milk, egg white, and a cell lysate were used as the samples to demonstrate the feasibility of this approach. In only 30 and 90 s, phosphopeptides and phosphoproteins sufficient for characterization by MALDI-MS were enriched by the particles, respectively. Proteins trapped on the particles could be directly digested on the particles. The same particles in the digest solution were employed for enrichment of phosphopeptides. We estimated the required time for performing the enrichment of phosphopeptides from complex samples and characterization by MALDI MS was within 5 min. A small volume (50 microL) and a low concentration (5 x 10(-10) M) of tryptic digest product of a phosphoprotein sample could be dramatically enriched and characterized using this approach.     

Analysis of protein phosphorylation by monolithic extraction columns based on poly(divinylbenzene) containing embedded titanium dioxide and zirconium dioxide nano-powders

The potential of an organic monolith with incorporated titanium dioxide (TiO(2)) and zirconium dioxide (ZrO(2)) nanoparticles was evaluated for the selective enrichment of phosphorylated peptides from tryptic digests. A pipette tip was fitted with a monolith based on divinylbenzene (DVB) of highly porous structure, which allows sample to pass through the monolithic bed. The enrichment of phosphopeptides was enhanced by increasing the pipetting cycles during the sample preparation and a higher recovery could be achieved with adequate buffer systems. A complete automated process was developed for enrichment of phosphopeptides leading to high reproducibility and resulting in a robust method designed to minimize analytical variance while providing high sensitivity at high sample throughput. The effect of particle size on the selectivity of phosphopeptides was investigated by comparative studies with nano- and microscale TiO(2) and ZrO(2) powders. Eleven phosphopeptides from alpha-casein digest could be recovered by an optimized mixture of microscale TiO(2)/ZrO(2) particles, whereas nine additional phosphopeptides could be retained by the same mixture of nano-structured material. When compared to conventional immobilized metal-ion affinity chromatography and commercial phosphorylation-enrichment kits, higher selectivity was observed in case of self fabricated tips. About 20 phosphopeptides could be retained from alpha-casein and five from beta-casein digests by using TiO(2) and ZrO(2) based extraction tips. Further selectivity for phosphopeptides was demonstrated by enriching a digest of in vitro phosphorylated extracellular signal regulated kinase 1 (ERK1). Two phosphorylated peptides of ERK1 could be identified by MALDI-MS/MS measurements and a following MASCOT database search.     

Preparation of C60‐functionalized magnetic silica microspheres for the enrichment of low‐concentration peptides and proteins for MALDI‐TOF MS analysis

In this work, for the first time, a novel C60‐functionalized magnetic silica microsphere (designated C60‐f‐MS) was synthesized by radical polymerization of C60 molecules on the surface of magnetic silica microspheres. The resulting C60‐f‐MS microsphere has magnetite core and thin C60 modified silica shell, which endow them with useful magnetic responsivity and surface affinity toward low‐concentration peptides and proteins. As a result of their excellent magnetic property, the synthesized C60‐f‐MS microspheres can be easily separated from sample solution without ultracentrifuge. The C60‐f‐MS microspheres were successfully applied to the enrichment of low‐concentration peptides in tryptic protein digest and human urine via a MALDI‐TOF MS analysis. Moreover, they were demonstrated to have enrichment efficiency for low‐concentration proteins. Due to the novel materials maintaining excellent magnetic properties and admirable adsorption, the process of enrichment and desalting is very fast (only 5 min), convenient and efficient. As it has been demonstrated in the study, newly developed fullerene‐derivatized magnetic silica materials are superior to those already available in the market. The facile and low‐cost synthesis as well as the convenient and efficient enrichment process of the novel C60‐f‐MS microspheres makes it a promising candidate for isolation of low‐concentration peptides and proteins even in complex biological samples such as serum, plasma, and urine or cell lysate.     

Enhancing the identification of phosphopeptides from putative basophilic kinase substrates using Ti (IV) based IMAC enrichment

Metal and metal oxide chelating-based phosphopeptide enrichment technologies provide powerful tools for the in-depth profiling of phosphoproteomes. One weakness inherent to current enrichment strategies is poor binding of phosphopeptides containing multiple basic residues. The problem is exacerbated when strong cation exchange (SCX) is used for pre-fractionation, as under low pH SCX conditions phosphorylated peptides with multiple basic residues elute with the bulk of the tryptic digest and therefore require more stringent enrichment. Here, we report a systematic evaluation of the characteristics of a novel phosphopeptide enrichment approach based on a combination of low pH SCX and Ti(4+)-immobilized metal ion affinity chromatography (IMAC) comparing it one-to-one with the well established low pH SCX-TiO(2) enrichment method. We also examined the effect of 1,1,1,3,3,3-hexafluoroisopropanol (HFP), trifluoroacetic acid (TFA), or 2,5-dihydroxybenzoic acid (DHB) in the loading buffer, as it has been hypothesized that high levels of TFA and the perfluorinated solvent HFP improve the enrichment of phosphopeptides containing multiple basic residues. We found that Ti(4+)-IMAC in combination with TFA in the loading buffer, outperformed all other methods tested, enabling the identification of around 5000 unique phosphopeptides containing multiple basic residues from 400 μg of a HeLa cell lysate digest. In comparison, ～ 2000 unique phosphopeptides could be identified by Ti(4+)-IMAC with HFP and close to 3000 by TiO(2). We confirmed, by motif analysis, the basic phosphopeptides enrich the number of putative basophilic kinases substrates. In addition, we performed an experiment using the SCX/Ti(4+)-IMAC methodology alongside the use of collision-induced dissociation (CID), higher energy collision induced dissociation (HCD) and electron transfer dissociation with supplementary activation (ETD) on considerably more complex sample, consisting of a total of 400 μg of triple dimethyl labeled MCF-7 digest. This analysis led to the identification of over 9,000 unique phosphorylation sites. The use of three peptide activation methods confirmed that ETD is best capable of sequencing multiply charged peptides. Collectively, our data show that the combination of SCX and Ti(4+)-IMAC is particularly advantageous for phosphopeptides with multiple basic residues.     

Highly robust, automated, and sensitive online TiO2-based phosphoproteomics applied to study endogenous phosphorylation in Drosophila melanogaster

Reversible protein phosphorylation ranks among the most important post-translational modifications, and elucidation of phosphorylation sites is essential to understand the regulation of key cellular processes such as signal transduction. Enrichment of phosphorylated peptides is a prerequisite for successful analysis due to their low stoichiometry, heterogeneity, and low abundance. Enrichment is often performed manually, which is inherently labor-intensive and a major hindrance in large-scale analyses. Automation of the enrichment method would vastly improve reproducibility and thereby facilitate 'high-throughput' phosphoproteomics research. Here, we describe a robust and automated online TiO 2-based two-dimensional chromatographic approach to selectively enrich phosphorylated peptides from digests of complete cellular lysates. We demonstrate method enhancement for both adsorption and desorption of phosphorylated peptides resulting in lower limits of detection. Phosphorylated peptides from a mere 500 attomole tryptic digest of a protein mixture were easily detected. With the combination of strong cation exchange chromatography with the online TiO 2 enrichment, 2152 phosphopeptides were enriched from 250 microg of protein originating for the cell lysate of Drosophila melanogaster S2 cells. This is a 4-fold improvement when compared to an enrichment strategy based solely on strong cation exchange/LC-MS. Phosphopeptide enrichment methods are intrinsically biased against relatively basic phosphopeptides. Analysis of the p I distributions of the enriched/detected phosphopeptides showed that the p I profile resembles that of a total Drosophila protein digest, revealing that the current described online procedure does not discriminate against either more acidic or basic phosphopeptides. However, careful comparison of our new and existing phosphopeptide enrichment techniques also reveal that, like many enrichment techniques, we are still far from comprehensive phosphoproteomics analyses, and we describe several factors that still require to be addressed. Still, as the online approach allows the complementary measurements of phosphopeptides and their nonphosphorylated counterparts in subsequent analyses, this method is well-suited for automated quantitative phosphoproteomics.     

Applications of monolithic silica capillary columns in proteomics

The use and applicability of silica based capillary monolithic reversed-phase columns in proteomic analysis has been evaluated by liquid chromatography-mass spectrometry (LC-MS). Chromatographic performance of the monolithic capillaries was evaluated with a tryptic digest of cytochrome C showing very good resolution and reproducibility in addition to the known advantages of a low pressure drop over a time period of 6 months. Monoliths were subsequently tested for their suitability to separate proteins and peptides from samples typically encountered in proteomic research such as in-gel digested tryptic peptide mixtures or fractions of proteolytically digested human serum. The monolithic capillaries also proved useful in the analysis of phospholipid species in bronchoalveolar lavage fluid. Compared to particle-filled conventional capillary columns, rapid and highly efficient separation of peptides and proteins was achieved using these bimodal pore size distribution columns, and good quality collision induced dissociation (CID) mass spectra were obtained on an ion trap mass spectrometer. These novel monolithic separation media are thus a promising addition to the methodological toolbox of proteomics research.     

Reduction of non-specific binding in Ga(III) immobilized metal affinity chromatography for phosphopeptides by using endoproteinase glu-C as the digestive enzyme

The selectivity of immobilized metal affinity chromatography (IMAC) systems for the purification of phosphopeptides is poor. This is particularly a problem with tryptic digests of proteins where a large number of acidic peptides are produced that also bind during IMAC. The hypothesis examined in this work was that the selectivity of IMAC columns for phosphopeptides could be increased by using endoproteinase glu-C (glu-C) for protein digestion. Glu-C cleaves proteins at acidic residues and should reduce the number of acidic residues in peptides. This method was successfully applied to a mixture of model proteins and bovine milk. The percentage of phosphorylated peptides selected from proteolytic digests of the milk sample was increased from 40% with trypsin to 70% with glu-C. Additionally, this method was coupled with stable isotope coding methods to quantitatively compare the concentration of phosphoproteins between samples.     

Enrichment of phosphorylated proteins and peptides from complex mixtures using metal oxide/hydroxide affinity chromatography (MOAC)

A novel method termed metal oxide affinity chromatography (MOAC) of enriching for phosphorylated proteins and peptides based on the affinity of the phosphate group for Al(OH)(3) is presented here. When compared to commercial phosphoprotein-enrichment kits, this method is more selective, more cost effective and easily applicable to method optimization. The use of glutamic and aspartic acid in the loading buffer significantly enhances selectivity. Standard protein mixtures and complex Arabidopsis thaliana leaf protein extracts were tested for efficacy of enrichment. The method can be applied to proteins extracted using either mild or denaturing conditions. The same Al(OH)(3) material is suitable for the enrichment of phosphopeptides out of a tryptic digest of alpha-casein. Peptide phosphorylation was revealed by beta-elimination of phosphate groups. Enrichment and in vivo phosphorylation of A. thaliana leaf proteins were confirmed with Pro-Q diamond stain. Several of the phosphoprotein candidates that were identified by MS are known to be phosphorylated in vivo in other plant species.     

A Novel Open Tubular Capillary Electrochromatographic Method for Differentiating the DNA Interaction Affinity of Environmental Contaminants

The interaction of chemicals with DNA may lead to genotoxicity, mutation or carcinogenicity. A simple open tubular capillary electrochromatographic method is proposed to rapidly assess the interaction affinity of three environmental contaminants (1,4-phenylenediamine, pyridine and 2,4-diaminotoluene) to DNA by measuring their retention in the capillaries coated with DNA probes. DNA oligonucleotide probes were immobilized on the inner wall of a fused silica capillary that was first derivatized with 3-(aminopropyl)-triethoxysilane (APTES). The difference in retention times and factors was considered as the difference in interaction affinity of the contaminants to the DNA probes. The interaction of the contaminants with both double-stranded (dsDNA) and single-stranded DNA (ssDNA) coatings was compared. Retention factors of 1,4-phenylenediamine, pyridine and 2,4-diaminotoluene in the capillary coated with ssDNA probe were 0.29, 0.42, and 0.44, respectively. A similar trend was observed in the capillary coated with dsDNA, indicating that 2,4-diaminotoluene has the highest affinity among the three contaminants. The relative standard deviation (RSD) for the retention factors was in the range of 0.05–0.69% (n = 3). The results demonstrated that the developed technique could be applied for preliminary screening purpose to provide DNA interaction affinity information of various environmental contaminants.     

Poly(glycidyl methacrylate/divinylbenzene)-IDA-FeIII in phosphoproteomics

The study of protein phosphorylation has grown exponentially in recent years, as it became evident that important cellular functions are regulated by phosphorylation and dephosphorylation of proteins on serine, threonine and tyrosine residues. The use of immobilized metal affinity chromatography (IMAC) to enrich phosphopeptides from peptide mixtures has been shown to be useful especially prior to mass spectrometric analysis. For the selective enrichment applying solid-phase extraction (SPE) of phosphorylated peptides, we introduce poly(glycidyl methacrylate/divinylbenzene) (GMD) derivatized with imino-diacetic acid (IDA) and bound Fe(III) as a material. GMD is rapidly synthesized and the resulting free epoxy groups enable an easy access to further derivatization with, e.g., IDA. Electron microscopy showed that the synthesized GMD-IDA-Fe(III) for SPE has irregular agglomerates of spherical particles. Inductively coupled plasma (ICP) analysis resulted in a metal capacity of Fe(III) being 25.4 micromol/mL. To enable on-line preconcentration and desalting in one single step, GMD-IDA-Fe(III) and Silica C18 were united in one cartridge. Methyl esterification (ME) of free carboxyl groups was carried out to prevent binding of nonphosphorylated peptides to the IMAC function. The recovery for a standard phosphopeptide using this SPE method was determined to be 92%. The suitability of the established system for the selective enrichment and analysis of model proteins phosphorylated at different amino acid residues was evaluated stepwise. After successful enrichment of beta-casein deriving phosphopeptides, the established system was extended to the analysis of in vitro phosphorylated proteins, e.g. deriving from glutathione-S-transferase tagged extracellular signal regulated kinase 2 (GST-ERK2).     

Zirconium phosphonate-modified porous silicon for highly specific capture of phosphopeptides and MALDI-TOF MS analysis

Phosphorylation is one of the most important post-translational modifications of proteins, which modulates a wide range of biological functions and activity of proteins. The analysis of phosphopeptides is still one of the most challenging tasks in proteomics research by mass spectrometry. In this study, a novel phosphopeptide enrichment approach based on the strong interaction of zirconium phosphonate (ZrP) modified surface with phosphopeptides has been developed. ZrP modified porous silicon (ZrP-pSi) wafer was prepared to specifically capture the phosphopeptides from complex peptide mixtures, and then the captured phosphopeptides were analyzed by MALDI-TOF MS by directly placing the wafer on a MALDI target. The phosphopeptide enrichment and MALDI analysis were both performed on the ZrP-pSi wafer which significantly reduced the sample loss and simplified the analytical procedures. The prepared ZrP-pSi wafer has been successfully applied for the enrichment of phosphopeptides from the tryptic digest of standard phosphoproteins beta-casein and alpha-casein. The excellent selectivity of this approach was demonstrated by analyzing phosphopeptides in the digest mixture of beta-casein and bovine serum albumin with molar ratio of 1:100. High detection sensitivity has been achieved for the analysis of the phosphopeptides from tryptic digestion of 2 fmol beta-casein on the ZrP-pSi surface.     

Development of an off-line capillary column IMAC phosphopeptide enrichment method for label-free phosphorylation relative quantification

Immobilized metal affinity chromatography (IMAC) and metal oxide type affinity chromatography (MOAC) techniques have been widely used for mass spectrometry-based phosphorylation analysis. Unlike MOAC techniques, IMAC requires rather complete removals of buffering reagents, salts and high concentrations of denaturant prior to sample loading in order for the successful enrichment of phosphopeptides. In this study, a simple off-line capillary column-based IMAC phosphopeptide enrichment method can shorten sample preparation time by eliminating the speed-vac step from the desalting process. Tryptic digest peptide samples containing 2M urea can be directly processed and the entire IMAC procedure can be completed within 6 h. When tryptic digest peptide samples prepared from mouse whole brain tissues were analyzed using our method, an average of 249 phosphoproteins and 463 unique phosphopeptides were identified from single 2-h RPLC-MS/MS analysis (~88% specificity). An additional advantage of this method is the significantly improved reproducibility of the phosphopeptide enrichment results. When four independent phosphopeptide enrichment experiments were carried out, the peak areas of phosphopeptides identified among four enrichment experiments were relatively similar (less than 16.2% relative standard dev.). Because of this increased reproducibility, relative phosphorylation quantification analysis of major phosphoproteins appears to be feasible without the need for stable isotope labeling techniques.     

Phosphoproteome profiling of human skin fibroblast cells in response to low- and high-dose irradiation

A hallmark of the response to high-dose radiation is the up-regulation and phosphorylation of proteins involved in cell cycle checkpoint control, DNA damage signaling, DNA repair, and apoptosis. Exposure of cells to low doses of radiation has well documented biological effects, but the underlying regulatory mechanisms are still poorly understood. The objective of this study is to provide an initial profile of the normal human skin fibroblast (HSF) phosphoproteome and explore potential differences between low- and high-dose irradiation responses at the protein phosphorylation level. Several techniques including Trizol extraction of proteins, methylation of tryptic peptides, enrichment of phosphopeptides with immobilized metal affinity chromatography (IMAC), nanoflow reversed-phase HPLC (nano-LC)/electrospray ionization, and tandem mass spectrometry were combined for analysis of the HSF cell phosphoproteome. Among 494 unique phosphopeptides, 232 were singly phosphorylated, while 262 peptides had multiple phosphorylation sites indicating the overall effectiveness of the IMAC technique to enrich both singly and multiply phosphorylated peptides. We observed approximately 1.9-fold and approximately 3.6-fold increases in the number of identified phosphopeptides in low-dose and high-dose samples respectively, suggesting both radiation levels stimulate cell signaling pathways. A 6-fold increase in the phosphorylation of cyclin dependent kinase (cdk) motifs was observed after low- dose irradiation, while high-dose irradiation stimulated phosphorylation of 3-phosphoinositide-dependent protein kinase-1 (PDK1) and AKT/RSK motifs 8.5- and 5.5-fold, respectively. High- dose radiation resulted in the increased phosphorylation of proteins involved in cell signaling pathways as well as apoptosis while low-dose and control phosphoproteins were broadly distributed among biological processes.