Specific phosphopeptide enrichment with immobilized titanium ion affinity chromatography adsorbent for phosphoproteome analysis

The elucidation of protein post-translational modifications, such as phosphorylation, remains a challenging analytical task for proteomic studies. Since many of the proteins targeted for phosphorylation are low in abundance and phosphorylation is typically substoichiometric, a prerequisite for their identification is the specific enrichment of phosphopeptide prior to mass spectrometric analysis. Here, we presented a new method termed as immobilized titanium ion affinity chromatography (Ti (4+)-IMAC) for enriching phosphopeptides. A phosphate polymer, which was prepared by direct polymerization of monomers containing phosphate groups, was applied to immobilize Ti (4+) through the chelating interaction between phosphate groups on the polymer and Ti (4+). The resulting Ti (4+)-IMAC resin specifically isolates phosphopeptides from a digest mixture of standard phosphoproteins and nonphosphoprotein (BSA) in a ratio as low as 1:500. Ti (4+)-IMAC was further applied for phosphoproteome analysis of mouse liver. We also compared Ti (4+)-IMAC to other enrichment methods including Fe (3+)-IMAC, Zr (4+)-IMAC, TiO 2 and ZrO 2, and demonstrate superior selectivity and efficiency of Ti (4+)-IMAC for the isolation and enrichment of phosphopeptides. The high specificity and efficiency of phosphopeptide enrichment by Ti (4+)-IMAC mainly resulted from the flexibility of immobilized titanium ion with spacer arm linked to polymer beads as well as the specific interaction between immobilized titanium ion and phosphate group on phosphopeptides.     

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.     

Quantitative proteomic analysis of mouse embryonic fibroblasts and induced pluripotent stem cells using 16O/18O labeling

Induced pluripotent stem cells (iPSC) hold great promise for regenerative medicine as well as for investigations into the pathogenesis and treatment of various diseases. Understanding of key intracellular signaling pathways and protein targets that control development of iPSC from somatic cells is essential for designing new approaches to improve reprogramming efficiency. Here, we report the development and application of an integrated quantitative proteomics platform for investigating differences in protein expressions between mouse embryonic fibroblasts (MEF) and MEF-derived iPSC. This platform consists of 16O/18O labeling, multidimensional peptide separation coupled with tandem mass spectrometry, and data analysis with UNiquant software. With this platform, a total of 2481 proteins were identified and quantified from the 16O/18O-labeled MEF-iPSC proteome mixtures with a false discovery rate of 0.01. Among them, 218 proteins were significantly upregulated, while 247 proteins were significantly downregulated in iPSC compared to MEF. Many nuclear proteins, including Hdac1, Dnmt1, Pcna, Ccnd1, Smarcc1, and subunits in DNA replication and RNA polymerase II complex, were found to be enhanced in iPSC. Protein network analysis revealed that Pcna functions as a hub orchestrating complicated mechanisms including DNA replication, epigenetic inheritance (Dnmt1), and chromatin remodeling (Smarcc1) to reprogram MEF and maintain stemness of iPSC.     

A quantitative analysis of Arabidopsis plasma membrane using trypsin-catalyzed (18)O labeling

Typical mass spectrometry-based protein lists from purified fractions are confounded by the absence of tools for evaluating contaminants. In this report, we compare the results of a standard survey experiment using an ion trap mass spectrometer with those obtained using dual isotope labeling and a Q-TOF mass spectrometer to quantify the degree of enrichment of proteins in purified subcellular fractions of Arabidopsis plasma membrane. Incorporation of a stable isotope, either H(2)(18)O or H(2)(16)O, during trypsinization allowed relative quantification of the degree of enrichment of proteins within membranes after phase partitioning with polyethylene glycol/dextran mixtures. The ratios allowed the quantification of 174 membrane-associated proteins with 70 showing plasma membrane enrichment equal to or greater than ATP-dependent proton pumps, canonical plasma membrane proteins. Enriched proteins included several hallmark plasma membrane proteins, such as H(+)-ATPases, aquaporins, receptor-like kinases, and various transporters, as well as a number of proteins with unknown functions. Most importantly, a comparison of the datasets from a sequencing "survey" analysis using the ion trap mass spectrometer with that from the quantitative dual isotope labeling ratio method indicates that as many as one-fourth of the putative survey identifications are biological contaminants rather than bona fide plasma membrane proteins.     

Purification of papain by immobilized metal affinity chromatography (IMAC) on chelating carboxymethyl cellulose

Chelating carboxymethyl cellulose was prepared in bead form by immobilizing iminodiacetic acid on carboxymethyl cellulose which was earlier crosslinked and activated by epichlorohydrin. The prepared matrix was used to purify papain by a factor of 2.6 from commercial papain, and by a factor of 4 from papaya latex by batch adsorption and immobilized metal affinity chromatography respectively. Purification factors obtained were equal in batch mode and double in column mode, to purifications obtained on Chelating Sepharose® Fast Flow. Flow rates up to 38 ml/cm2 h were easily possible on the prepared chelating carboxymethyl cellulose.     

Effects of docosahexaenoic acid on mouse brain synaptic plasma membrane proteome analyzed by mass spectrometry and (16)O/(18)O labeling

Docosahexenoic acid (DHA, 22:6n-3) plays an important role in development of proper brain function in mammals. We have previously reported that DHA promotes synaptogenesis and synaptic function in hippocampal neurons while DHA-depletion in the brain due to n-3 fatty acid deficiency produces opposite effects. To gain insight into underlying molecular mechanisms, we investigated whether the brain DHA status affects the synaptic plasma membrane (SPM) proteome by using nanoLC-ESI-MS/MS and (16)O/(18)O labeling. The DHA level in mouse brains was lowered by dietary depletion of n-3 fatty acids, and SPM was prepared by differential centrifugation followed by osmotic shock. SPM proteins from DHA-adequate and depleted brains were analyzed by nanoLC-ESI-MS/MS after SDS-PAGE, in-gel digestion, and differential O(18)/O(16) labeling. This strategy allowed comparative quantitation of more than 200 distinct membrane or membrane-associated proteins from DHA-adequate or depleted brains. We found that 18 pre- and postsynaptic proteins that are relevant to synaptic physiology were significantly down-regulated in DHA-depleted mouse brains. The protein network analysis suggests involvement of CREB and caspase-3 pathways in the DHA-dependent modulation of synaptic proteome. Reduction of specific synaptic proteins due to brain DHA-depletion may be an important mechanism for the suboptimal brain function associated with n-3 fatty acid deficiency.     

Purification of recombinant histidine-tag streptolysin O using immobilized metal affinity expanded bed adsorption (IMA-EBA)

In this report, we describe the recombinant SLO expression as a fusion protein with a C-terminal hexahistidine tag and its purification using immobilized metal affinity expanded bed adsorption (STREAMLINE(trade mark) Chelating). In order to facilitate downstream processing of the purification, an efficient fermentation process was developed focusing on the achievement of high yields of soluble protein. The purification strategy resulted in a 40% recovery of active recombinant SLO and the protein was purified eight-fold. SDS-PAGE and Western-blot analysis of the purified protein revealed the presence of a 75 Mr form, which was the estimated relative Mass of the recombinant SLO.     

Enrichment and analysis of nonenzymatically glycated peptides: boronate affinity chromatography coupled with electron-transfer dissociation mass spectrometry

Nonenzymatic glycation of peptides and proteins by d-glucose has important implications in the pathogenesis of diabetes mellitus, particularly in the development of diabetic complications. However, no effective high-throughput methods exist for identifying proteins containing this low-abundance post-translational modification in bottom-up proteomic studies. In this report, phenylboronate affinity chromatography was used in a two-step enrichment scheme to selectively isolate first glycated proteins and then glycated, tryptic peptides from human serum glycated in vitro. Enriched peptides were subsequently analyzed by alternating electron-transfer dissociation (ETD) and collision induced dissociation (CID) tandem mass spectrometry. ETD fragmentation mode permitted identification of a significantly higher number of glycated peptides (87.6% of all identified peptides) versus CID mode (17.0% of all identified peptides), when utilizing enrichment on first the protein and then the peptide level. This study illustrates that phenylboronate affinity chromatography coupled with LC-MS/MS and using ETD as the fragmentation mode is an efficient approach for analysis of glycated proteins and may have broad application in studies of diabetes mellitus.     

Purification of unique alpha subunits of GTP-binding regulatory proteins (G proteins) by affinity chromatography with immobilized beta gamma subunits

Novel G protein alpha subunits were purified from rat brain by an affinity matrix containing immobilized beta gamma subunits (Pang, I.-H., and Sternweis, P. C. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 7814-7818). They were unique based on the following criteria. These alpha subunits migrated differently through polyacrylamide gels with an apparent molecular mass of 42 kDa. They did not behave similarly to the other brain G proteins by conventional chromatographic techniques. Antisera raised against a common region of known alpha subunits failed to recognize these 42-kDa polypeptides. Finally, primary sequences of tryptic fragments of these proteins contain regions homologous to, yet unique from, the other alpha subunits. Sequences are identical with one or more members of a new family of alpha subunits recently identified by molecular genetic techniques (Strathmann, M., Wilke, T. M., and Simon, M. I. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 7407-7409); most of the primary sequence identifies an alpha subunit labeled alpha q. These polypeptides were not substrates for ADP-ribosylation catalyzed by pertussis toxin. They bound GTP gamma S only with slow rates and low stoichiometry. Antisera to peptides based on primary sequence were specific for the new alpha subunits and indicate that they are widely distributed at low levels in different tissues but more concentrated in brain and lung. This procedure provides a means of preparing native G proteins that have a potential role as modulators of pertussis toxin-insensitive regulatory pathways.     

Quantitative analysis of monofluoroacetate in biological samples by high-performance liquid chromatography using fluorescence labeling with 9-chloromethylanthracene

A rapid and sensitive RP-HPLC method with fluorescence detection has been developed for the quantitative analysis of trace amounts of monofluoroacetate (MFA) in biological samples as serum, food and meat. 9-Chloromethylanthracene (9-CMA) is used as the fluorescence labeling reagent. Samples were extracted and reacted with 9-chloromethylanthracene together with tetrabutylammonium bromide as catalyst at 80 degrees C for 50 min to give a new fluorescent derivative as 9-methyleneanthracene monofluoroacetate (MA-MFA). The resulting MA-MFA was characterized with IR, (1)H NMR, (13)C NMR and MS. Chromatography separation is performed on an Agilent Hypersil ODS column with a fluorescent detector employed with the excitation and emission wavelengths as 256 nm and 412 nm, respectively. Optimal conditions for derivatization, fluorescence detection and chromatographic separation have been established. The novel method yields a good linear relationship when the MFA concentration in serum within 1 and 250 ng/mL (r=0.9988). The detection limit (signal-to-noise ratio=3 with 2 microL injected) was 0.25 ng/mL. The practical applicability of this method was demonstrated by quantitative determination of MFA-Na in a blood sample from a person who had ingested the poison.     

Quantitative proteome analysis of human plasma following in vivo lipopolysaccharide administration using 16O/18O labeling and the accurate mass and time tag approach

Identification of novel diagnostic or therapeutic biomarkers from human blood plasma would benefit significantly from quantitative measurements of the proteome constituents over a range of physiological conditions. Herein we describe an initial demonstration of proteome-wide quantitative analysis of human plasma. The approach utilizes postdigestion trypsin-catalyzed 16O/18O peptide labeling, two-dimensional LC-FTICR mass spectrometry, and the accurate mass and time (AMT) tag strategy to identify and quantify peptides/proteins from complex samples. A peptide accurate mass and LC elution time AMT tag data base was initially generated using MS/MS following extensive multidimensional LC separations to provide the basis for subsequent peptide identifications. The AMT tag data base contains >8,000 putative identified peptides, providing 938 confident plasma protein identifications. The quantitative approach was applied without depletion of high abundance proteins for comparative analyses of plasma samples from an individual prior to and 9 h after lipopolysaccharide (LPS) administration. Accurate quantification of changes in protein abundance was demonstrated by both 1:1 labeling of control plasma and the comparison between the plasma samples following LPS administration. A total of 429 distinct plasma proteins were quantified from the comparative analyses, and the protein abundances for 25 proteins, including several known inflammatory response mediators, were observed to change significantly following LPS administration.     

Simultaneous determination of lipoic acid (LA) and dihydrolipoic acid (DHLA) in human plasma using high-performance liquid chromatography coupled with electrochemical detection

A fast, simple, and a reliable high-performance liquid chromatography linked with electrochemical detector (HPLC-ECD) method for the assessment of lipoic acid (LA) and dihydrolipoic acid (DHLA) in plasma was developed using naproxen sodium as an internal standard (IS) and validated according to standard guidelines. Extraction of both analytes and IS from plasma (250 μl) was carried out with a single step liquid-liquid extraction applying dichloromethane. The separated organic layer was dried under stream of nitrogen at 40°C and the residue was reconstituted with the mobile phase. Complete separation of both compounds and IS at 30°C on Discovery HS C18 RP column (250 mm × 4.6 mm, 5 μm) was achieved in 9 min using acetonitrile: 0.05 M phosphate buffer (pH 2.4 adjusted with phosphoric acid) (52:48, v/v) as a mobile phase pumped at flow rate of 1.5 ml min(-1) using electrochemical detector in DC mode at the detector potential of 1.0 V. The limit of detection and limit of quantification for lipoic acid were 500 pg/ml and 3 ng/ml, and for dihydrolipoic acid were 3 ng/ml and 10 ng/ml, respectively. The absolute recoveries of lipoic acid and dihydrolipoic acid determined on three nominal concentrations were in the range of 93.40-97.06, and 93.00-97.10, respectively. Similarly coefficient of variations (% CV) for both intra-day and inter-day were between 0.829 and 3.097% for lipoic acid and between 1.620 and 5.681% for dihydrolipoic acid, respectively. This validated method was applied for the analysis of lipoic acid/dihydrolipoic acid in the plasma of human volunteers and will be used for the quantification of these compounds in patients with oxidative stress induced pathologies.     

Enrichment of phosphoproteins for proteomic analysis using immobilized Fe(III)-affinity adsorption chromatography

We described an efficient protocol to strongly enrich phosphoproteins from mixtures of total cellular proteins using homemade, recyclable Fe(III)-affinity columns. An integral feature of the method is the use of a detergent cocktail that allows use of different pHs for total protein extraction (pH 6.8) and for subsequent affinity capture of phosphoproteins (pH 3.4). Affinity captured proteins from rat fibroblasts were fractionated on 2D gels and random selection was identified by mass spectrometry. More than 85% of identified proteins were previously known to be phosphorylated. The specificity of the method was further validated by isolating proteins from (32)P labeled cells. Our comparison of the clusters of acidic residues in the captured proteins with acidic clusters in proteins of the rat genome indicates that affinity for phosphate groups dominates over adsorption of proteins with acidic clusters.     

Evaluation of the interaction of peptides with Cu(II), Ni(II), and Zn(II) by high-performance immobilized metal ion affinity chromatography

High-performance immobilized metal ion affinity chromatography was utilized to evaluate the adsorption properties of 67 synthetic, biologically active, peptides ranging in size from 5 to 42 residues. The metal ions, Cu(II), Ni(II) and Zn(II), were immobilized by iminodiacetic acid (IDA) coupled to TSK gel 5PW (10 microns). Two types of gradient elution (imidazole and pH) were used to evaluate peptide retention by the metal ions. A decreasing pH gradient and an increasing imidazole gradient eluted the peptides in similar order. IDA-Cu(II) and IDA-Zn(II) showed very similar selectivities for the peptides analyzed; however, IDA-Zn(II) displayed a weaker affinity for the peptides. IDA-Ni(II) showed a slightly different pattern of selectivity. Peptide adsorption effects contributed by the metal-free gel matrix were found to be relatively minor. The concentration and type of salt included in the mobile phase could affect the relative affinities of the peptides for the immobilized metal ions. Retention coefficients were assigned to individual amino acid residues by multiple linear regression analysis. Histidine showed the largest positive correlation with retention, followed by aromatic amino acid residues. Modified N-terminal residues resulted in negative contributions to retention. Analyses of peptide amino acid composition alone allowed prediction of peptide retention behavior on immobilized metal ion affinity columns.     

Purification and characterization of two types of Cytisus sessilifolius anti-H(O) lectins by affinity chromatography.

Two anti-H(O) lectins were separated from extracts of Cytisus sessilifolius seeds by successive affinity chromatographies on columns of di-N-acetylchitobiose- and galactose-Sepharose 4B. One was found to be inhibited most by di-N-acetylchitotriose or tri-N-acetylchitotriose [Cytisus-type anti-H(O) lectin designated as Cytisus sessilifolius lectin I (CSA-I)] and the other anti-H(O) lectin was inhibited by galactose or lactose and designated as Cytisus sessilifolius lectin II (CSA-II). These two anti-H(O) lectins were further purified by gel filtration on TSK-Gel G3000SW. These preparations were homogeneous as judged by polyacrylamide gel electrophoresis and gel filtration. The molecular masses of the purified lectins I and II were found to be 95,000 and 68,000 Da, respectively, by gel filtration on TSK-Gel G3000SW. On polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and 2-mercaptoethanol, both lectins gave a single component of molecular masses of 27,000 +/- 2,000 and 34,000 +/- 2,000 Da, respectively, suggesting that the lectins I and II were composed of four and two apparently identical subunits, respectively. Lectins I and II contain 38% and 13% carbohydrate, respectively, and only very small amounts of cysteine and methionine, but they are rich in aspartic acid, serine and glycine. The N-terminal amino-acid sequences of these two lectins were determined and compared with those of several lectins already published.     

Photoaffinity labeling of high affinity nicotinic acid adenine dinucleotide phosphate (NAADP)-binding proteins in sea urchin egg

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a messenger that regulates calcium release from intracellular acidic stores. Recent studies have identified two-pore channels (TPCs) as endolysosomal channels that are regulated by NAADP; however, the nature of the NAADP receptor binding site is unknown. To further study NAADP binding sites, we have synthesized and characterized [(32)P-5-azido]nicotinic acid adenine dinucleotide phosphate ([(32)P-5N(3)]NAADP) as a photoaffinity probe. Photolysis of sea urchin egg homogenates preincubated with [(32)P-5N(3)]NAADP resulted in specific labeling of 45-, 40-, and 30-kDa proteins, which was prevented by inclusion of nanomolar concentrations of unlabeled NAADP or 5N(3)-NAADP, but not by micromolar concentrations of structurally related nucleotides such as NAD, nicotinic acid adenine dinucleotide, nicotinamide mononucleotide, nicotinic acid, or nicotinamide. [(32)P-5N(3)]NAADP binding was saturable and displayed high affinity (K(d) ～10 nM) in both binding and photolabeling experiments. [(32)P-5N(3)]NAADP photolabeling was irreversible in a high K(+) buffer, a hallmark feature of NAADP binding in the egg system. The proteins photolabeled by [(32)P-5N(3)]NAADP have molecular masses smaller than the sea urchin TPCs, and antibodies to TPCs do not detect any immunoreactivity that comigrates with either the 45-kDa or the 40-kDa photolabeled proteins. Interestingly, antibodies to TPC1 and TPC3 were able to immunoprecipitate a small fraction of the 45- and 40-kDa photolabeled proteins, suggesting that these proteins associate with TPCs. These data suggest that high affinity NAADP binding sites are distinct from TPCs.     

A study of glycoproteins in human serum and plasma reference standards (HUPO) using multilectin affinity chromatography coupled with RPLC-MS/MS

The glycoproteome is a major subproteome present in human plasma. In this study, we isolated and characterized approximately 150 glycoproteins from the human plasma and serum samples provided by HUPO using a multilectin affinity column. The corresponding tryptic digest was separated by RP-HPLC coupled to an IT mass spectrometer (3-D LCQ). Also in this study, a new system, namely an Ettan MDLC system coupled to a linear ITLTQ, was compared with the previous LCQ platform and gave a greater number of protein identifications, as well as better quality. When we compared the composition of the glycoproteomes for the plasma and serum samples there was a close correlation between the samples, except for the absence of fibrinogen from the identified-protein list in the latter sample, which was presumably as a result of the clotting process. In addition, the analysis of the samples from three ethnic specimens, Caucasian American, Asian American, and African American, were very similar but showed a higher angiotensinogen plasma level and a lower histidine-rich glycoprotein level in Caucasian American samples, and a lower vitronectin level in African American blood samples.     

Quantitative proteomic analysis of membrane proteins involved in astroglial differentiation of neural stem cells by SILAC labeling coupled with LC-MS/MS

Membrane proteins play a critical role in the process of neural stem cell self-renewal and differentiation. Here, we apply the SILAC (stable isotope labeling by amino acids in cell culture) approach to quantitatively compare the membrane proteome of the self-renewing and the astroglial differentiating cells. High-resolution analysis on a linear ion trap-Orbitrap instrument (LTQ-Orbitrap) at sub-ppm mass accuracy resulted in confident identification and quantitation of more than 700 distinct membrane proteins during the astroglial differentiation. Of the 735 quantified proteins, seven cell surface proteins display significantly higher expression levels in the undifferentiated state membrane compared to astroglial differentiating membrane. One cell surface protein transferrin receptor protein 1 may serve as a new candidate for NSCs surface markers. Functional clustering of differentially expressed proteins by Ingenuity Pathway Analysis revealed that most of overexpressed membrane proteins in the astroglial differentiation neural stem cells are involved in cellular growth, nervous system development, and energy metabolic pathway. Taken together, this study increases our understanding of the underlying mechanisms that modulate complex biological processes of neural stem cell proliferation and differentiation.