Article pubs.acs.org/ac
Widening and Diversifying the Proteome Capture by Combinatorial Peptide Ligand Libraries via Alcian Blue Dye Binding Giovanni Candiano,†,⊥ Laura Santucci,†,⊥ Andrea Petretto,‡ Chiara Lavarello,‡ Elvira Inglese,‡ Maurizio Bruschi,† Gian Marco Ghiggeri,† Egisto Boschetti,§ and Pier Giorgio Righetti*,∥ †
Nephrology, Dialysis, Transplantation Unit and Laboratory on Pathophysiology of Uremia, Istituto Giannina Gaslini, Genoa 16148, Italy ‡ Core FacilitiesProteomics Laboratory, Istituto Giannina Gaslini, Genoa 16148, Italy § EB JAM-Conseil, 92200 Neuilly sur-Seine, Paris, France ∥ Department of Chemistry, Materials and Chemical Engineering, “Giulio Natta”, Politecnico di Milano, Via Mancinelli 7, Milano 20131, Italy S Supporting Information *
ABSTRACT: Combinatorial peptide ligand libraries (CPLLs) tend to bind complex molecules such as dyes due to their aromatic, heterocyclic, hydrophobic, and ionic nature that may affect the protein capture specificity. In this experimental work Alcian Blue 8GX, a positively charged phthalocyanine dye well-known to bind to glycoproteins and to glucosaminoglycans, was adsorbed on a chemically modified CPLL solid phase, and the behavior of the resulting conjugate was then investigated. The control and dye-adsorbed beads were used to harvest the human urinary proteome at physiological pH, this resulting in a grand total of 1151 gene products identified after the capture. Although the Alcian Blue-modified CPLL incremented the total protein capture by 115 species, it particularly enriched some families among the harvested proteins, such as glycoproteins and nucleotidebinding proteins. This study teaches that it is possible, via the two combined harvest mechanisms, to drive the CPLL capture toward the enrichment of specific protein categories. hen first introduced in 2005,1,2 the methodology of combinatorial peptide ligand libraries (CPLLs) exhibited immediately a unique capability in digging much deeper into any proteome and revealing a large variety of lowabundance species that would normally escape detection by any current method, including the most advanced mass spectrometry equipment. It was understood that this was due to its unique mechanism of action, enabling the simultaneous reduction of high-abundance proteins (HAP) while enriching for the low-abundance ones (LAP) via a continuous harvesting of such scarce species from solution. The enhancement of visibility of LAPs could reach, in favorable cases and according to the sample availability, up to 3−4 orders of magnitude.3 To further improve the efficiency of such libraries, modifications to the initial protocols have been proposed. A succinylated variant has been described,4 and the capture step has been enlarged from pH 4 to pH 9 with an extended protein harvesting.5 A reduction of the ionic strength during the capture process contributed also to improved results as reported by Di Girolamo et al.6 Another important improvement has been focused on a more complete protein elution as described by Candiano et al.,7 in the presence of 4% sodium dodecyl sulfate (SDS) added with 3% dithioerythrol (DTE).
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© XXXX American Chemical Society
In attempting at driving the capture toward a more hydrophobic class of compounds, yet another variant has been introduced in the presence of high concentrations of lyotropic salts of the Hofmeister series (1 M ammonium sulfate) favoring hydrophobic interaction.8 As a result 28% of captured proteins were additionally found only because of “hydrophobically” driven interaction.9 A recent review reports all these advances in the attempt to maximize the efficiency of the technology.10 It is within this context that modifiers such as transition metal ions could be adsorbed on CPLLs prior to protein capture, thus affecting the collection of different proteins. In order to ascertain if this principle can be generalized to other modifiers, we have investigated the effect of adsorption of Alcian Blue to the hexapeptides of the CPLL beads, with some interesting results here reported.
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MATERIALS AND METHODS Chemicals. The solid-phase combinatorial peptide library (ProteoMiner), protein staining and glycoprotein staining
Received: January 9, 2015 Accepted: April 9, 2015
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Figure 1. Chemical structure of Alcian Blue 8GX (on the right) and its docking on the carboxylated peptide beads represented on the left. The dye structure comprises four cationic groups (inset on the upper right) covalently attached on the aromatic rings. These structures facilitate the adsorption on the peptide terminal groups by ion exchange leaving thus exposed the planar part of the dye comprising four isoindols with their aromatic rings (in red) and heterocycles trapping in the middle an atom of a transition metal that is, in this case, copper.
adsorption and during the protein elution phase. The presence of low concentration of EDTA in the sample upon loading did not seem having any stripping effect on copper ions that are strongly coordinated on the dye. Sample Treatment with the Solid-Phase Ligand Library. The 17 000g urinary supernatant (10 mg of proteins) was treated with 150 μL of peptide library beads in different modes, as follows: either with the succinylated form of ProteoMiner or the same with adsorbed Alcian Blue dye. In all cases, samples and beads were equilibrated in 25 mM phosphate buffer, pH 7.2, added with 12 mM sodium azide. An amount of 5 mM EDTA was also added to the urine sample as antibacterial and for metal protease inhibition activity. Elution was performed in 4% boiling SDS, 3% DTT as per Candiano et al.7 After removal of SDS via acetone/acid precipitation,12 all eluates were subjected to 2D electrophoresis and mass spectrometry analysis. Two-Dimensional (2D) Gel Electrophoresis. The pellets of dry proteins were dissolved and lyophilized as previously described13 and then globally analyzed by 2D electrophoresis according to Bruschi et al.14 A glycoprotein specific detection was performed in parallel with the same samples. The staining was performed using Pro-Q Emerald 488 solution following the recommendation of the supplier. The images were digitalized by means of FX Pro laser scanner (BioRad). Mass Spectrometry Analysis. The sample preparation (2 μg), the nanoLC setup, and mass spectrometer setup were tasks performed exactly as described in Santucci et al.;15 data analysis was also performed according to the same referenced paper. To facilitate the removal of undesired materials such as detergents, filters with cutoff of 30 kDa have been used as reported by Wisniewski et al.16 who demonstrated that relatively small unfolded peptides can efficiently be retained by quite large cutoffs.
solutions for electrophoresis, and gel plaques and gel reagents were from Bio-Rad (Hercules, CA, U.S.A.). The PD-Quest software analysis program vs8.3, VersaDoc 4000 and Molecular Imager GS-800 calibration densitometer were purchased from BioRad. Alcian Blue 8GX, succinic anhydride, urea, thiourea, 3(3-cholamidopropyl dimethylammonio)-1-propansulfonate (CHAPS), DTE, tris(hydroxymethyl)aminomethane, ethylendiamino tetracetic acid (EDTA), and sodium dodecyl sulfate were all from Sigma-Aldrich (St. Louis, MO, U.S.A.). Complete protease inhibitor cocktail tablets were from Roche Diagnostics (Basel, CH). Sequencing grade bovine trypsin was from Promega (Madison, WI, U.S.A.). All other chemicals of analytical grade were from J.T. Baker (Deventer, Holland). Urine Collection and Preparation. Second morning urines (approximately 160 mL), from healthy volunteers (six individuals, age 35−45 years, three males and three females) were collected after informed consent. Urines were immediately added with tablets of protease inhibitor, chilled on ice and centrifuged at 4 °C for 10 min at 1000g to eliminate cell debris. The urines were further centrifuged at 17 000g in a JA-20 rotor (Beckman Avanti J-25) for 30 min at 16 °C to remove the urinary microscopic particle sediments. All urine samples were processed fresh. An aliquot of the total 17 000g urinary supernatant, after Bradford protein assay, was dialyzed three times against 25 mM sodium phosphate pH 7.2 in 3500 MWCO Spectra/Por cellulose membranes at 4 °C. This material was stored at −80 °C until use. Preparation of Alcian Blue−CPLL Conjugate. Before the adsorption of Alcian Blue 8GX on CPLL beads, the latter were chemically transformed by succinylation according to a protocol previously described in all details.4,11 Succinylated CPLL beads were extensively washed with 25 mM phosphate buffer pH 7.2. In parallel a solution of Alcian Blue was prepared in the same buffer at a concentration of 1 mg/mL. An amount of 100 μL of Alcian Blue solution was added to 100 μL of succinylated CPLL beads, and the suspension gently shaken overnight at room temperature. The excess of dye was eliminated by centrifugation at 1000g for 10 min, and the beads extensively washed with phosphate buffer. The collected stained beads appeared stable when exposed to the sample
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RESULTS AND DISCUSSION Alcian Blue 8GX is a cationic dye that does not naturally adsorb on native CPLLs unless they are succinylated, as already reported. 1 7, 1 8 The structure of this dye [tetrakisB
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Figure 2. Upper panel: two-dimensional gel electrophoresis (2-DE) pattern comparison of urinary proteins collected from the control succinylated CPLL resin (A) or the same sample treated with the succinylated CPLL resin saturated with Alcian Blue (B). The nonlinear IPG gradient was between pH 3 and 10. Protein detection was with silver stain. Lower panel: Venn diagram of the total proteins (1151) detected in urines treated with succinylated beads (gray circle) and the same saturated with Alcian Blue (black circle). The graph shows the exclusive enrichment of proteins by both the succinylated CPLL resin saturated with Alcian Blue and the control succinylated CPLL.
regions as well as the four isoindol-like heterocyclic structures surrounding an atom of copper. It is with this solid phase that urinary proteins have been captured and then collected for the analyses of the proteome composition. Figure 2 (upper panel) shows the comparative 2D maps of control, succinylated CPLLs, versus the same beads saturated with Alcian Blue, both captures performed at pH 7.2. It can be appreciated that the patterns share a large number of protein spots located throughout the 2D map. However, it is remarkable to see a quite enhanced capture by the Alcian Blue beads of many species within the 10−20 kDa region, probably fragments of larger proteins suggesting that the Alcian Blue structure modulates the mechanism of protein interaction. High molecular mass proteins are also found; this could appear surprising since the filtration through kidney glomeruli suggests that no protein larger than about 50 kDa should be present. However, the presence of large species such as ankyrin, dynein heavy chain, cation-independent mannose-6-phosphate receptor, plectin, and many others has been observed in normal urines and published in various old20,21 and recent papers.2,22,23 A possible explanation is the release of proteins that are stripped from tissues after the glomeruli filtration. A similar analysis focused on glycoproteins was made by using a specific staining (Supplementary Figure 1 of the Supporting Information). It shows that, in addition to proteins found in the control, a large number of additional glycoproteins were found upon sample treatment with dye−bead conjugate demonstrating the expected propensity of the dye to capture more glycoproteins. Since two-dimensional electrophoresis neither reveals the name of the exclusive protein spots, nor their function and composition, mass spectrometry analyses have been performed
(tetramethylisothiouronium) copper phthalocyanine tetrachloride], as shown in Figure 1, is a planar large organic molecule capable of displaying various interactions with biological molecules. It consists of four geometrical isomers substituted at position 4 of each isoindole residue, and it comprises a copper ion complexed within the nitrogen of heterocyclic moieties. It is a well-known cationic phthalocyanine dye adopted in histology for staining negatively charged mucopolysaccharides, such as heparin, and titrating the total number of sulfate and carboxyl groups along its backbone.19 In its classical use at pH above 3−4 this dye interacts with carboxyls of uronic and sialic acids present in mucins and acidic glycoproteins. Its use under more acidic conditions allows a strong interaction with glucosaminoglycans due to the presence of sulfonates groups. In its native free form this planar dye is relatively hydrophilic due both to the presence of heterocycles and to the symmetric positioning of four quite strong cationic groups that are well-placed to interact with uronic acids. The resulting interaction with proteins likely results from a dominant ionexchange effect. However, the presence of heterocycles and of the atom of copper also modulates the docking mechanisms. In addition this dye comprises four hydrophobic aromatic rings that presumably play a role in the interaction with proteins. Once this dye is exposed to succinylated CPLLs, ionic interactions occur between the amino groups of the dye and the flexible carboxyls at the terminal end of the grafted hexapeptides, thus favoring a stable docking. In fact no dye adsorption was observed on native CPLLs (which expose primary amino groups) unless they are succinylated. Under this configuration the dye is very tightly conjugated with the peptides and turns the beads from white to light blue, thus exposing its planar surface comprising aromatic hydrophobic C
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Analytical Chemistry and compared with the control (succinylated CPLL without Alcian Blue). The overall protein harvesting was very substantial with 1151 unique gene products as illustrated in Figure 2 (lower panel) by means of Venn diagrams and as seen in the list of Supplementary Table 1 in the Supporting Information. Interestingly it is observed that in spite of a large protein overlap (876 gene products or 76% of total) a significant number of proteins were exclusively captured by Alcian Blue−CPLL resin: 115 gene products (see Supplementary Table 2 in the Supporting Information). Also noticeable is the fact that the control experiment captured exclusively 160 gene products that were rejected by the phthalocyanine dye highlighting the involvement of the adsorbed dye on the beads prior the protein capture step. Actually the affinity constant of proteins captured by ligand libraries are very broad from strong affinity to very weak interaction; a little modification of the hexapeptide ligands (here is the presence of Alcian Blue) can result in an enhanced or in a decreased affinity constant driving thus the capture of the species toward the one or the other type of beads. The large number of common proteins (about 76%) is explained by the still present dominant role of the hexapeptides that is only slightly modulated by the dye. Nevertheless, Alcian Blue contributed to add novel urinary gene products never described before (see Figure 3 and Supplementary Table 2 in the
Supporting Information). Actually, when comparing to the current extensive knowledge on urinary proteome several observations can be made. The overall number of proteins known up to now is of 4430 gene products with a major contribution by Santucci et al.15 corresponding to 3275 unique proteins. Other important contributions can be seen from Marimuthu et al.22 with 1823 species, followed by Adachi et al.23 with 1543 units, Li et al.24 with 1205 proteins and by Zerefos et al.25 with 559 gene products. Naturally, when comparing these studies a large number of proteins are superimposed to each other, and overall the total known number becomes 4430. As illustrated, the use of Alcian Blue associated with succinylated CPLLs added another set of novel exclusive proteins never described before in prior urinary proteomes, which is quite modest (38 unique and exclusive new proteins in the blue area), but because of the very large number of species already described, one could not expect to find many more additional proteins. It is here interesting to note that a number of proteins from the present workeven if modest in numberhave no equivalents in previous studies, all others being massively present within the findings of Santucci et al.15 and the other authors reported above.22−25 This situation is not really surprising when one thinks of the use of phthalocyanine dyes as ligands for affinity chromatography. A similar phthalocyanine dye ligand (Reactive Blue 15, also called Procion Turquoise MXG) was successfully used for the separation of proteins such as yeast alcohol dehydrogenase26 and oxalate oxidase.27 Interestingly, more recently, phthalocyanine dyes have been described as binders for multiple sites on natively folded prion protein with a potential impact as antiscrapie agents to prevent the formation of the infectious, misfolded prion protein.28 From mass spectrometry findings the physicochemical and functional characteristics of the harvested proteins have been classified on Supplementary Table 1 in the Supporting Information. Among the specific data assembled and compared with the control beads are isoelectric points and hydrophobic Gravy index. The association of these values for each protein is illustrated in Figure 4, where the blue dots represent all species exclusively captured by the dye−bead conjugate and the red dots by the control beads. Although the whole composite map does not immediately evidence the hydrophobic enhanced property of the dye beads, the global calculation of Gravy indexes of each protein from Alcian Blue beads shows a quite significant increase of the average value (about 10%) compared to proteins captured exclusively by the control succinylated beads. Reproducibility of all these data has been evaluated by the principal component analysis (PCA) and the Spearman’s coefficient (R2) correlogram. Data are illustrated on Supplementary Figure 2, parts A and B, in the Supporting Information, respectively, where two experimental clusters are evidenced (control and Alcian Blue beads). A good reproducibility was confirmed by the near overlap of the six samples and the high R2 of each experiment. These R2 values were in the range of 0.81−0.96 and 0.84−0.97 for control and CPLL with adsorbed dye, respectively. The degree of biological variation due to the CPLL process was defined as a coefficient of variation (CV) of 0.25 for the control and 0.23 for dye−beads conjugate. From a gene ontology analysis, as illustrated in Figure 5, specific categories of proteins emerged such as “carbohydrate binding proteins” and “nucleotide binding proteins” suggesting that the presence of Alcian Blue dye induces a quite complex
Figure 3. Venn diagram comprising proteins from different bibliographic sources compared to proteins found in the present work. The blue surface area refers to Alcian Blue−beads captured proteins from the present work (total 991 gene products, 115 exclusive of the dye− bead conjugate and 38 never published before). Only urinary proteins that were never disclosed before are represented since all others are “drowned” within all other contribution and impossible to be visualized. The pink surface refers to gene products found by Santucci et al. (ref 15) with a total of 3275 unique proteins. The green area encompasses data from Marimuthu et al. (ref 22) with 1823 unique proteins. The gray area comprises data from Adachi et al. (ref 23) with 1397 gene products (none are exclusive, all of them being described by the other authors). The yellow area from Li et al. (ref 24) represents an overall of 1166 gene products. The brown area collects 559 unique proteins described by Zerefos et al. (ref 25), the majority of them being exclusive with 413 units. D
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Figure 4. Composite map crossing over pI values and Gravy index values of proteins exclusively captured by Alcian Blue peptide beads (blue dots) and control succinylated peptide beads (red dots). The hydrophobic intensity of proteins increases in moving up from negative numbers to positive ones. The isoelectric point of proteins increases from left to right.
aggregates even in the presence of some nonpolar solvents as a result of their planar configuration.29 These molecules display hydrophobic properties and form π−π conjugated complexes that are quite stable even when repulsing charges are present. Globally these molecules are slightly hydrophobic, and to render them more compatible with aqueous buffers they have been modified by the introduction of either sulfonated groups or cationic groups, as illustrated in Figure 1. In the present case it is hypothesized that the extra set of proteins exclusively captured by the dye might be due to hydrophobic association favored by the fact that the four positively charged groups of the dye are neutralized via binding to the peptide carboxyl termini. This is influenced by the exposed aromatic rings of the dye that are part of the four indol-like structures. Indeed the phthalocyanine molecules have hydrophobic properties responsible for the association with lipid bilayers;30 thus, some hydrophobic associations with proteins are expected. Glycoprotein Capture Enhancement by Alcian Blue− CPLL Beads. Strong lectin-like properties of Alcian Blue have been shown by François et al.31 when attempting to prevent infections of immunodeficiency viruses such as HIV-1 and HIV2. It has been demonstrated that Alcian Blue selectively inhibits retrovirus infection, by binding to the glycans of the gp120 envelope. The latter is highly glycosylated and clearly binds to the dye in spite of the fact that the N-glycans comprising a large amount of high mannose are neither charged nor preferentially recognized by Alcian Blue. In the present study we found that many of the captured gene products by the Alcian Blue resin are glycoproteins. In the past decade indol-like structures have been described as capable of interacting with glycans. Tryptamine and aminoindan have actually been reported as important structures for the design of ligands for the recognition of glycoproteins.32,33 It seems that such structures are capable to surround the sugar molecules and form hydrogen bonds between the planar polar nitrogen groups
Figure 5. Pie charts of the main molecular function GO assigned to the proteins identified from the exclusive Alcian Blue beads capture. The main constituents are nucleotide-binding proteins (30%), catalytic activity proteins (16%), cation-binding proteins (16%), and carbohydrate-binding proteins (9%).
mixed-mode interaction behavior. Among the identified protein those belonging to either “carbohydrate binding” or “nucleotide binding” are highlighted in Supplementary Table 2 in the Supporting Information. Several hypotheses could thus be formulated on the dominant nature of the novel-captured proteins: (i) the dye is modified in its hydrophilic−hydrophobic balance with an increased propensity to capture hydrophobic proteins; (ii) the dye still shows some capability to associate with acidic glycoproteins; (iii) the neutralized dye remains planar and displays heterocycles with conjugated double bonds that could also induce peculiar interactions with nucleotide-binding proteins. Proteins Captured by the Enhanced Hydrophobic Property of the Dye−CPLL Conjugate. Phthalocyanines are scarcely soluble in water and are prone to form stacked E
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examining some of the proteins of the Alcian Blue exclusive list it appears that several of them have clinical interest and could now be detected in urines. This is the case of guanine nucleotide-binding protein (GBP) that is described as being radioresistant; hence cancer patients with expressed GBP tend to have a poor response to radiotherapy.41 Another example is given by DNA damage-binding protein that interacts not only with DNA but also with small mimetic molecules such as cyclobutane pyrimidine dimers and is purified by a chromatographic sequence where Cibacron Blue plays a key role with its mixed-mode mimetic interaction.42 Both proteins are relatively hydrophobic as indicated on Supplementary Tables 1 and 2 in the Supporting Information, thus confirming that the interaction with the dye is not dependent on the presence of ionic charges. Possible Influence of the Copper Ion Carried by the Phthalocyanine Structure. The phthalocyanine structure used comprises a copper atom that provides coordination sites to interact with some amino acids of proteins. This is the principle exploited in metal ion affinity chromatography, also called IMAC.43 However, this metal ion is strongly associated with the dye organic structure and it is unclear how it can influence the protein capture. In spite of that the functional GO analysis shows that the cation binding proteins increased compared to the control. This group includes cadherin,44 phosphopantothenate-cysteine ligase45 as well as zinc finger protein,46 all of them purified by means of IMAC. Other metal ions such as cobalt, zinc, nickel, and aluminum can form complexes in the place of copper with some influence on protein interaction. For instance, zinc-complexed phthalocyanine was used to induce interaction with the homodimeric protein HP7.29 In another example copper and nickel have been compared for their ability to bind human telomere Gquadruplex with quite similar dissociation constants.47 These specific points are yet to be elucidated and are part of our ongoing investigations.
and the equatorial 3- and 4-hydroxyl groups of the saccharides. This molecular interaction is displayed toward neutral hexoses in the order mannose > glucose > galactose. From these ligands it has been demonstrated that it was possible to discriminate between glycoproteins and nonglycosylated proteins. Hence, glucose oxidase as oligomannose-type model has thus been successfully separated. Other glycoproteins were also adsorbed on such ligands such as peroxidase, ovalbumin, kallikrein, invertase, fibrinogen, and α1-acidic glycoprotein that were selectively eluted by means of methyl-β-D-mannoside. A refinement of these structures comprising indol-like molecules has been recently described by Chen et al.34 The ligand structure derived from the multicomponent Ugi reaction involves four compounds, where one of them is tryptamine, a molecule very similar to the four units of phthalocyanine of the present work. The resulting ligand was also very effective in capturing glycoproteins with the following sugar specificity preference: ribose > arabinose > xylose > galactose > mannose > glucose. With this in mind we examined the properties of the species eluted from the blue beads and found that indeed a number of the exclusively captured proteins by the Alcian Blue beads are glycoproteins. This is the case for instance of sialoadhesin, a myelin-associated glycoprotein found in the list of Alcian Blue exclusive proteins, which is a large mass protein having cystein-rich regions as lectins with distinct sugar epitopes depending on the cells where from is expressed. The glycans comprise multiantenna structures with several sugars and a certain degree of sulfation.35 N-Acetyl-D-glucosamine kinase is also a glycoprotein that, beyond its acidic character that allows its adsorption on DEAE resins, also strongly adsorbs onto Phenyl Sepharose as described,36 demonstrating a dominant hydrophobic character with a Gravy index +0.113 (see Table 2 on the Supporting Information provided). Another example is the choline transporter-like protein, an inner ear glycoprotein with multiple N-glycosylation sites that is highly hydrophobic (its Gravy index is +0.327).37 Other examples of proteins exclusively found in dye−beads conjugate that are hydrophobic and glycosylated are alanine aminotransferase, inositol monophosphatase, and 6-phosphofructokinase. For all of them the calculated isoelectric point, that appears slightly basic, with, respectively, 7.2, 8.2, 7.54, does not necessarily fit with the reality since their value is obtained from the amino acid composition only. Nucleotide-Binding Gene Products Enhancement by Alcian Blue Resin. The classification of captured proteins under a functional GO shows a majority of them involved in nucleotide binding (see Figure 5 and Supplementary Table 2 in the Supporting Information). It is unclear why this is the case; nevertheless, when looking at the structure of the phthalocyanine, it is possible to find a certain degree of molecular similarities to nucleotidic structures especially guanine where a number of conjugated nitrogens are equally present. Although this hypothesis is speculative it cannot be discarded when a certain degree of mimetic behavior is assumed. Actually in 2008 Zhang et al.38 described phthalocyanines as potential inhibitors of telomerase due to their ability to stabilize G-quadruplex, a four-stranded DNA structure with stacked guanine tetrads, as recently confirmed.39 G-quadruplexes formed by different guanine-rich sequences are found in genomic DNA and interestingly are also generated during in vitro screening for aptamers targeting proteins.40 In this situation it is assumed that phthalocyanine could also behave as a nucleotide mimetic with the capability to bind or enhance protein binding. When
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CONCLUSION The pattern of captured proteins by means of peptide ligand library modified by Alcian Blue is altered as a result of the modulation of the affinity adsorption, which in turns is dependent on the multiple features brought by the dye itself. The latter contributes to mixed-mode interaction and produces the enhancement of several categories of proteins. Several situations are easily explained and could be anticipated such as the additional specificity for glycoproteins; others are discovered afterward. The protein capture by the hexapeptide ligands is not dramatically revolutionized with the adsorption of Alcian Blue dye; however, this is a way to address specific categories of proteins opening thus novel opportunities to be further explored. The peculiar behavior of the investigated dye could be extended to other structures that could be adsorbed onto CPLLs. The door is here open for a large number of exploration opportunities.
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ASSOCIATED CONTENT
S Supporting Information *
Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. F
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G.C. and L.S. contributed equally to this work and share first authorship. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
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REFERENCES
The Giannina Gaslini Institute provided financial and logistical support to this study. This work was also supported by the Italian Ministry of Health “Ricerca Corrente” and from contributions derived from “Cinque per mille dell’IRPEF”. We also acknowledge contributions from the Renal Child Foundation, Fondazione Mara Wilma e Bianca Querci (project “Ruolo dello stress reticolare nella progressione del danno renale e tumorale”), Fondazione La Nuova Speranza (“Progetto integrato per la definizione dei meccanismi implicati nella glomerulo sclerosi focale”), and Italian Society of Nephrology (Progetto Ricercando).
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DOI: 10.1021/acs.analchem.5b00218 Anal. Chem. XXXX, XXX, XXX−XXX
