In-Depth Exploration of Cow's Whey Proteome via Combinatorial

In-Depth Exploration of Cow’s Whey Proteome via Combinatorial Peptide Ligand Libraries Alfonsina D’Amato,† Angela Bachi,† Elisa Fasoli,‡ Egisto Boschetti,§ Gabriel Peltre,| Hele`ne Se´ne´chal,| and Pier Giorgio Righetti*,‡ San Raffaele Scientific Institute, 20132 Milano, Italy, Department of Chemistry, Materials and Chemical Engineering “Giulio Natta”, Politecnico di Milano, 20131 Milano, Italy, Bio Rad Laboratories, C/o CEA-Saclay, 91181 Gif-sur-Yvette, France, and Laboratoire Environnement et Chimie Analytique, UMR CNRS 7121, ESPCI, Paris, France Received March 6, 2009

The use of combinatorial peptide ligand libraries, containing hexapeptides terminating with a primary amine, or modified with a terminal carboxyl group, allowed discovering and identifying a large number of previously unreported proteins in cow’s whey. Whereas comprehensive whey protein lists progressively increased in the last 6 years from 17 unique gene products to more than 100, our findings have considerably expanded this list to a total of 149 unique protein species, of which 100 were not described in previous proteomics studies. As an additional interesting result, a polymorphic alkaline protein was observed with a strong positive signal when blotted from an isoelectric focusing separation in gel and tested with sera of allergic patients. This polymorphic protein, found only after treatment with the peptide library, was identified as an immunoglobulin (Ig), a minor allergen that had been largely amplified. The list of cow’s whey components here reported is by far the most comprehensive at present and could serve as a starting point for the functional characterization of low-abundance proteins possibly having novel pharmaceutical, diagnostic, and biomedical applications. Keywords: peptide ligand libraries • low-abundance proteome • cow’s milk • allergens • mass spectrometry • proteomics

Introduction Mature bovine milk contains about 3.3% protein, of which about 80% consists of caseins (CN), namely, RS1-, RS2-, β- and κ-caseins, the remaining 20% being serum albumin, β-lactoglobulin, R-lactalbumin and other low-abundance proteins.1 Milk is the most important food for young mammals and a common source of proteins and microelements for adult people. In addition, it is an important means for transferral of immunity to pathogens from the mother to the newborn, as it contains antimicrobial and immunomodulatory proteins that are active in the digestive tract of newborns. Cow’s milk is of great human nutritional and economic significance, yet its repertoire of minor proteins has not been characterized in depth. The first proteomic analysis of cow’s and human milk, perhaps, goes back to a two-dimensional (2D) mapping study of Anderson et al.,2 who identified just a handful of proteins, in fact the seven most abundant proteins listed above. In more recent times, Hamdan’s group3,4 has explored the content of bovine milk either powdered or fresh after 2D mapping, spot excision and identification via MALDI-TOF mass * To whom correspondence should be addressed. Fax: +39 02 23993080. E-mail: [email protected]. † San Raffaele Scientific Institute. ‡ Politecnico di Milano. § Bio Rad Laboratories. | Laboratoire Environnement et Chimie Analytique. 10.1021/pr900221x CCC: $40.75

 2009 American Chemical Society

spectrometry analysis. Although only the seven major proteins could be detected, several post-translational modifications (PTM) could be assessed, such as up to five phosphorylations in caseins and lactose adducts in R-lactoglobulin. The problem of identifying low-abundance proteins (LAP) in bovine colostral and mature milk was first addressed by Yamada et al.,5 who, for that purpose, first immuno-depleted their samples via antibodies against β-casein and immunoglobulin G (IgG). Even with this precaution, the results were meagre: a total of only 15 unique gene products could be described in both types of milk. More recent research focused on the bovine milk fat globule membrane (MFGM) proteome,6 as isolated from the floating milk fat: they identified 120 unique gene proteins in their membranaceous material, of which 71% were membraneassociated proteins, with the remainder being cytoplasmic or secreted proteins. In another approach, when trying to characterize host defense proteins in bovine milk, Smolenski et al.7 reported a total of 95 distinct gene products as collected findings in three fractions, namely, skim milk, whey and MFGMs. By the same token, in the case of water buffalo, D’Ambrosio et al.,8 by analyzing skim milk, whey and MFGMs, via 2D MALDI-TOF MS and electrospray-MS/MS analysis of trypsinized slices of SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), could count a total of 72 unique gene products. Journal of Proteome Research 2009, 8, 3925–3936 3925 Published on Web 06/05/2009

research articles In a parallel research line, a number of reports have appeared in proteome analysis of human colostrum and adult milk. In a 2D map analysis of MFGM, Giuffrida’s group9,10 has isolated 107 spots, which have been reduced to a total of 39 unique gene products. In a final review article,11 this group has listed a total of 73 unique gene products, as the sum of total findings in colostrum, mature milk, whey and MFGMs. Perhaps the most extensive findings reported so far are those of Palmer et al.,12 who, in the exploration of the low-abundance proteins in the aqueous phase of human colostrum, after immunoadsorption of immunoglobulin A, lactoferrin, R-lactalbumin and serum albumin, have listed a grand total of 151 unique gene products, of which 83 had not been previously reported. Whereas the aqueous phase of human colostrum has been mapped to a considerable extent,12 it is in a way disappointing that in the report of Smolenski et al.7 considerably fewer proteins have been detected, considering that, out of the total of 95 unique species reported, 55 are present only in the MFGM fraction, with another 28 common to two or more fractions, and only 17 found specifically in the whey, as opposed to the 151 species in colostrum.12 Considering that milk should contain low levels of serum-derived proteins, as well as proteins secreted from neutrophils and lymphocytes that have infiltrated the mammary gland, in addition to proteins derived from shedding and lysis of somatic cells,7 it is hard to accept that a total finding of 17 minor proteins in whey should represent the full cartography of the whey proteome. In an attempt at exploring to a much deeper extent the whey proteome, we have applied to this search the technique of combinatorial peptide ligand library (CPLL), that has worked with outstanding results in a number of other proteomic investigations, as reported in several recent reviews.13-20 As stated above, milk is an important human nutrient especially for young children; however, it is not exempt from specific drawbacks. Actually, milk is not always well tolerated by young patients since a number of allergy situations are encountered. The major milk allergens, those which are the most frequently recognized by patients IgE antibodies, are the different caseins, R-lactalbumin and β-lactoglobulin. Minor allergens such as lactoferrin, serum albumin and immunoglobulins are however known. These minor allergens are difficult to detect in ELISA and blotting techniques using whole milk, maybe due to antibody competition on overlapping antigens, particularly due to caseins, high-abundance species that extend throughout the migration lanes of SDS-PAGE and isoelectric focusing (IEF) analyses. Only immunodetections performed after removal of potential allergens that hide the signal of others allow detecting most subtle allergens generally present in trace amounts. Thus, real estimates of the sensitization rate of patients by these minor allergens are not known. Combinatorial peptide ligand libraries with their peculiar properties of enhancing low-abundance species are here evaluated with milk proteins to try solving this issue. Two-levels of IEF in gel followed by immunoblotting were performed with unambiguous detection of polymorphic immunoglobulins. In the present report, we give a detailed description of the process and its ability to find proteins that would otherwise be impossible to detect.

Materials and Methods Materials. Commercially available pasteurized whole milk with trade name of Marguerite was bought in a supermarket. The solid-phase combinatorial peptide Library-1 (ProteoMiner), 3926

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D’Amato et al. and its carboxylated version (Library-2, noncommercially available), as well as materials for electrophoresis such as gel plaques and reagents were from Bio-Rad Laboratories (Hercules, CA). N-ethylmaleimide, urea, thiourea, 3-[3-cholamidopropyl dimethylammonio]-1-propansulfonate (CHAPS), isopropanol, acetonitrile, trifluoroacetic acid and sodium dodecyl sulfate were all from Sigma-Aldrich (St Louis, MO). Complete protease inhibitor cocktail tablets were from Roche Diagnostics, (Basel, Switzerland). Sequencing grade porcine trypsin was from Promega (Madison, WI). Polyacrylamide gel (CleanGel) IEF, carrier ampholyte pH 3-10 and Multiphor II chamber, were from GE Healthcare, Uppsala, Sweden. Isoelectric point markers from pI 3 to 10 were from Serva, Heidelberg, Germany. Optitran BA-S 83 nitrocellulose membrane was from Schleicher & Schuell, Dassel, Germany. Immobilon-P, a PVDF blotting membrane with a porosity of 0.2 µM, was supplied by Millipore, Billerica, MA. Alkaline phosphatase-conjugated goat antihuman IgE, 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and 61.2 mM nitroblue tetrazolium (NBT) were purchased from Sigma-Aldrich, Saint Louis, MO. All other chemical were also from Sigma-Aldrich and were of analytical grade. Sample Collection and ProteoMiner Treatment. Five hundred milliliters of adult cow’s milk was centrifuged (2000g, 20 min) at 4 °C and the fat layer (with the milk fat globules) was carefully removed. The fat-free solution was then ultracentrifuged (100 000g, 1 h) so as to separate the casein micelles. The clear supernatant (whey fraction) was added with the Roche complete protease inhibitor tables and stored frozen at -20 °C until further use. To 500 mL of whey (5 g of total protein), 12 mM dry monopotassium phosphate was added along with 75 mM dry sodium chloride so as to get the equivalent of PBS; the pH was checked to 7.2. The solubilization of salts directly to the whey instead of adding PBS in solution allowed minimizing protein dilution. The sample thus obtained was mixed with 1 mL of ProteoMiner (Library-1) and shaken 7 h at room temperature. Library-1 was washed twice with PBS to remove excess soluble proteins and the captured proteins were eluted with TUC solution (2 M thiourea, 7 M urea, and 2% CHAPS) to which 25 mM cysteic acid was added (final pH 3.18). The supernatant from Library-1 was then mixed with 1 mL of Library-2 and again gently shaken for 7 h. After filtration, Library-2 was washed twice with PBS to remove nonadsorbed proteins and the captured species were eluted according to the same procedure used for Library-1. The two eluates were immediately neutralized, submitted to protein content analysis by the Bradford-Lowry standard spectrophotometric method, desalted by dialysis at 4 °C against 35% ethanol (cutoff of dialysis membrane was 1000 Da), and lyophilized. 1D-SDS-PAGE Analysis. Ten microliters of each sample (corresponding to ca. 30 µg of protein) was mixed with 10 µL of Laemmli buffer21 (4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.004% bromophenol blue, and 0.125 M Tris HCl, pH approximately 6.8). The mixture was heated in boiling water for 5 min and immediately loaded in the gel. The SDS-PAGE slab was composed by a stacking gel (125 mM Tris-HCl, pH 6.8, 0.1% SDS) with a large pore polyacrylamide (4%) cast over the resolving gel (8-18% acrylamide gradient in 375 mM TrisHCl, pH 8.8, 0.1% SDS buffer). The cathodic and anodic compartments were filled with Tris-glycine buffer, pH 8.3, containing 0.1% SDS. Electrophoresis was at 100 V until the dye front reached the bottom of the gel. Staining and destaining were performed with Colloidal Coomassie Blue21 and 7% acetic acid in water, respectively. The SDS-PAGE gels were scanned

Exploration of Cow’s Whey Proteome with a Versa-Doc image system (Bio-Rad Laboratories). For MS analysis, 60-120 µg of total proteins were loaded per track; at the end of the run, 11 slices were excised that covered the whole gel resolving region. 2D-PAGE Analysis. The desired volume of each nontreated sample and eluates was solubilized in the “2-D sample buffer” (7 M urea, 2 M thiourea, 3% CHAPS, and 40 mM Tris) to a final concentration of 2 mg/mL protein and the disulfide bridge reduction allowed to proceed at room temperature for 60 min by addition of TCEP [Tris(2-carboxyethyl)phosphine hydrochloride] at final concentration of 5 mM. For alkylating reduced -SH groups, 150 mM De-Streak [Bis-(2-hydroxyethyl)disulfide, (HOCH2CH2)2S2] (diluted directly from the stock 8.175 M, Sigma-Aldrich) was added to the solution, followed by 0.5% Ampholine (diluted directly from the stock, 40% solution) and a trace amount of bromophenol blue. Seven-centimeters long IPG strips (Bio-Rad), pH 3-10 L, were rehydrated with 150 µL of protein solution, for 4 h. IEF was carried out with a Protean IEF Cell (Bio-Rad Laboratories) in a linear voltage gradient from 100 to 1000 V for 5 h, 1000 V for 4 h, followed by an exponential gradient up to 5000 V, for a total of 25 kV/h. For the second dimension, the IPG strips were equilibrated for 25 min in a solution containing 6 M urea, 2% SDS, 20% glycerol, 375 mM Tris-HCl (pH 8.8) under gentle shaking. The IPG strips were then laid on 8-18% acrylamide gradient SDS-PAGE gel slab with 0.5% agarose in the cathodic buffer (192 mM glycine, 0.1% SDS and Tris-HCl to pH 8.3).22 The electrophoretic run was at 5 mA/gel for 1 h, followed by 10 mA/gel for 1 h and 15 mA/gel until the dye front reached the gel bottom. Gels were incubated in a colloidal Coomassie Blue solution and destaining was performed in 7% acetic acid until clear background, followed by a rinse in pure water. The 2-DE gels were scanned with a Versa-Doc image system (Bio-Rad), by fixing the acquisition time at 10 s; the relative gel images were captured via the PDQuest software (Bio-Rad Laboratories). After filtering the gel images for removing the background, spots were automatically detected, manually edited, and then counted. Protein Identification by NanoLC-MS/MS. The various sample lanes of SDS-PAGE gels were cut in 11 pieces of about 0.5 cm along the migration path, and proteins were reduced by 10 mM DTT and alkylated by 55 mM iodoacetamide. The gel pieces were shrunk in acetonitrile and dried under vacuum; proteins were digested overnight with trypsin as described elsewhere.23 The tryptic mixtures were acidified with formic acid up to a final concentration of 1%. Five microliters of tryptic digest for each band was injected in a capillary chromatographic system (EasyLC, Proxeon Biosystem, Denmark). Peptide separations occurred on a RP homemade 10-cm reverse phase spraying fused silica capillary column (75 µm i.d. × 10 cm), packed with 3-µm ReproSil 100C18 (Dr. Maisch GmbH, Germany). A gradient of eluents A (distilled water with 2% (v/v) acetonitrile, 0.1% (v/v) formic acid) and B (acetonitrile with 2% (v/v) distilled water with 0.1% (v/v) formic acid) was used to achieve separation, from 8% B (at 0 min 0.2 µL/min flow rate) to 50% B (at 80 min, 0.2 µL/min flow rate). The LC system was connected to an LTQ-Orbitrap mass spectrometer (ThermoScientific, Bremen, Germany) equipped with a nanoelectrospray ion source (Proxeon Biosystems, Odense, Denmark). Full scan mass spectra were acquired in the LTQ Orbitrap mass spectrometer in the mass range m/z 350 to 1500 Da and with the resolution set to 60 000. For accurate mass measurements, the lock-mass option was used.24 The four most intense doubly and triply charged ions were automatically selected and

research articles fragmented in the ion trap. Target ions already selected for the MS/MS were dynamically excluded for 60 s. All experiments were performed in triplicate. Data Analysis. The data were searched against the full International Protein Index (IPI) database with Mascot search engine (Matrix Science, London, U.K., version 2.1.04). We used tryptic cleavage constraints with maximum of 2 missed cleavages, cysteine alkylation and oxidation of methionine residues as variable modification, peptide mass tolerance was set to 5 ppm and fragment mass tolerance to 0.8 Da. The criteria used for protein identification were 20 ion score cutoff, protein Mascot score more than 100 and at least 2 unique peptides sequenced. Scaffold (version Scaffold-01_06_07, Proteome Software, Inc., Portland, OR) was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 95.0% probability as specified by the Peptide Prophet algorithm.25 Protein identifications were accepted if they could be established at greater than 99.0% probability and contained at least 2 identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm. Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. Allergen Detection by IEF and Immunoblotting. Freshly prepared whey from pasteurized whole milk previously defatted by centrifugation was submitted to an IEF separation performed in a polyacrylamide gel (CleanGel IEF) rehydrated into 5% (v/v) carrier ampholyte pH 3-10 in distilled water. Ten microliters of milk for each cm of gel was added to the gel surface on lines 1 cm apart from and parallel to the anode. The flat bed electrophoretic Multiphor II chamber was cooled at 15 °C. After the migration, a side strip of the gel was Coomassie blue stained. The gel plate was blotted by pressure for 1 h at 22 °C onto a wet Optitran BA-S 83 nitrocellulose membrane, covered by one dry sheet of Whatman filter paper, a glass plate and a 1 kg weight. The blotted membrane was dried to fix proteins on its surface, rewetted, and blocked with phosphate buffered saline pH 7.4 (PBS) containing 0.3% (v/v) Tween 20 (PBS-Tw) for 1 h at 22 °C. The membrane was cut into 26 narrow strips, each one incubated overnight at room temperature with a 10× PBS-diluted allergic patient serum. The selection of the child patient sera was based on the presence of specific IgE against whole milk generating severe allergic reactions. Samples were collected as left over from other clinical analyses. The strips were washed 4 times in PBS-Tw and incubated with alkaline phosphatase-conjugated goat antihuman IgE (1:700 in PBS-Tw) for 1 h at room temperature. The strips were washed again 4 times in PBS-Tw. The alkaline phosphatase activity was detected using 115.2 mM 5-bromo4-chloro-3-indolyl phosphate (BCIP) and 61.2 mM nitroblue tetrazolium (NBT) in 0.1 mol · L-1 Tris-HCl buffer, pH 9.5. Allergens from the whole milk, used as control, eluates from Library-1 and Library-2 and the flow-through were analyzed by immunoblot from an IEF polyacrylamide gel. The procedure was the same as described above except that this gel was rehydrated overnight in TU (2 M thiourea, 7 M urea), since the library eluates were obtained in TUC (2 M thiourea, 7 M urea, 2% CHAPS). CHAPS was omitted in order to avoid saturation of the blotting membrane during the protein transfer on it. The blotting membrane was a PVDF membrane with a porosity of 0.2 µM, which explains the differences in background and in enzymatic staining observed. All samples were Journal of Proteome Research • Vol. 8, No. 8, 2009 3927

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Figure 1. SDS-PAGE of Ctrl ) whey starting sample; FT ) Flowthrough after Library-1 and Library-2 treatments; Lib ) eluate (7 M urea, 2 M thiourea, 2% CHAPS and 25 mM cysteic acid, pH ) 3.18) after ProteoMiner treatment; Lib-2 ) eluate (7 M urea, 2 M thiourea, 2% CHAPS and 25 mM cysteic acid, pH ) 3.18) after Library-2 treatment. All samples loaded in 15 µL volume (30 µg of protein concentration). The broken horizontal lines represent the slicing positioning with slice number, prior to protein extraction, digestion and LC-MS/MS analysis. Staining with colloidal Coomassie blue.

at the same protein concentration of 50 mg/mL. Ten microliters of sample was applied for each cm of gel onto a 5 mm wide blotting paper strip laid on the gel surface on each lane, 1 cm apart from and parallel to the anode.

Results We briefly recall here that the combinatorial peptide ligand library is a mixture of porous beads on which hexapeptides are chemically attached. When a complex protein extract is exposed to such a ligand library under large overloading conditions, each bead with affinity to an abundant protein will rapidly become saturated, and the vast majority of the same protein will remain unbound. In contrast, trace proteins will not saturate the corresponding partner beads, but are captured in progressively increasing amounts as the beads are loaded with additional protein extract. After washing out excess of proteins, all captured species are desorbed from the beads: all proteins are present, but in a reduced dynamic concentration range dependent on the level of overloading conditions. Thus, a solid-phase ligand library enriches for trace proteins, while concomitantly reducing the relative concentration of abundant species. In this paper, we used combinatorial peptide ligand libraries to milk whey in order to try finding rare or very dilute protein species. Among them, we highlighted the possibility of potentially revealing low-abundance allergens. The initial milk selected for this work was pasteurized whole milk because all sera from milk-allergic patients were always screened for their IgE content to allergens on milk whey of most “native” form of commercial milk (namely, pasteurized whole bovine milk). Alternative methods for the preparation of whey may induce differences in the concentration of certain proteins species. Figure 1 shows the SDS-PAGE profiling of the control whey (Ctrl), of the two mixed flow-through (FT) solutions from both types of CPLLs and of the two TUC-cysteic acid eluates from 3928


D’Amato et al. the standard ProteoMiner (Lib-1) and its carboxylated version (Lib-2). As expected, there are no visible differences between Ctrl and FT samples, since, even after library treatments, only the major protein species, that have saturated the CPLLs, emerge unscathed, the minor components being not quite visible in both types of samples. Conversely, after peptide library treatments, as the major species concentration is drastically cut and the minor ones are concentrated onto the ligands, an almost continuum of bands appears in the entire Mr interval (10-250 kDa). However, with monodimensional electrophoresis, differences between eluates from the two libraries were not all clearly detected. Beyond the first impression of complementarity given by SDS-PAGE, the major aim of this analysis was to slice migration lanes in view of further mass spectrometry analysis for gene product identification. More exhaustive protein patterns of each fraction are given by two-dimensional electrophoresis. Figure 2 shows the 2D mapping of the starting material (Ctrl, upper panel), of the Library-1 eluate (lower left panel), followed by the Library-2 eluate (lower right panel). By visual inspection of the various panels, two phenomena are immediately apparent: the eluates from the two distinct libraries contain many more spots compared to the control, especially in the 10-50 kDa region. In addition, the Library-2 eluate seems to be somewhat complementary to the ProteoMiner capture, in that it appears to contain quite a few more alkaline species, which are absent in the Library-1 eluate. This is not surprising, considering that the extra negative charge at the hexapeptide terminus could preferentially bind higher pI compounds, as opposed to the positive charge present in the amino terminus standard ProteoMiner. The three samples (Ctrl, Library-1 and Library-2 eluates) were then loaded onto an SDS-PAGE slab, separated electrophoretically and stained; then 11 slices, that covered the whole gel resolving region, were excised. Each gel slice was subjected to trypsin digestion and the extracted peptides were analyzed by nanoLC-MS/MS in an Orbitrap instrument. The total findings are displayed in Table 1, which lists a total of 149 unique gene products, a far cry form the 17 species found in whey by Smolenski et al.7 or the 33 proteins listed in water buffalo’s whey by D’Ambrosio et al.8 Figure 3A gives overlapping Venn diagrams showing the distribution of detected proteins in the three samples, those unique to each sample and those shared among them. By comparison of the list of proteins found in the present study and the largest list of published milk whey gene products,12 100 proteins could be exclusively found in the present study as illustrated in Figure 3B. A number of proteins detected by Palmer et al.12 were found in the Ctrl (38 out of 149 or 25%); 11 other gene products were found either in the eluate of Library-1 or of Library-2. This is a quite large number when considering that many of the proteins identified by Palmer et al.12 were present only in the MFGM fraction. Among the 49 common proteins found, 78% were present in the Ctrl, which indicates that most, if not all of them, are quite high abundance and detectable without specific treatments. The difference between proteins found in common could be explained by the difference in equipment sensitivity (dissimilar mass spectrometers were used). Of the proteins that were not shared with the selected literature (100), 16 were exclusively found in the Ctrl (16%), 55 were exclusively from Library-1 and/or Library-2 (55%). All others (29%) were shared between Ctrl and the libraries. Table 2 reports the lists of

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Figure 2. Two-dimensional maps of Ctrl ) whey starting sample (upper panel); Lib-1 ) eluate (7 M urea, 2 M thiourea, 2% CHAPS and 25 mM cysteic acid, pH ) 3.18) after ProteoMiner treatment (lower left panel); Lib-2 ) eluate (7 M urea, 2 M thiourea, 2% CHAPS and 25 mM cysteic acid, pH ) 3.18) after Library-2 treatment (lower right panel). All samples adsorbed onto the IPG pH 3-10 strips in 150 µL volume (200 µg of protein concentration). Staining with colloidal Coomassie blue.

proteins exclusively captured by the Library-1 and the Library-2 that were absent from the selected literature. Interestingly, Library-2 allowed capturing almost as many as Library-1 proteins that would have otherwise escaped the detection of low-abundance proteins. Figure 4 illustrates the functional ontology classification of all proteins found in either control (nontreated initial whey, Figure 4A) and in library merged eluates (Figure 4B). Independently from the abundance of protein categories, there are modifications of protein category ranking, illustrating the general enhancement of proteins that were of low-abundance prior to library treatment with a concomitant decrease of categories that were largely represented. Since bovine milk contains known allergens, a specific preliminary analysis was performed to check the possible presence of allergenic antigens that generally are difficult to detect directly. Actually, with the use of ProteoMiner beads, high-abundance species are decreased in concentration, while some level of amplification of very low-abundance proteins is induced with consequent increased probability to find active allergenic species present in trace amount. Eluates from the two combinatorial libraries were first separated by isoelectric focusing (see Materials and Methods) and then blotted with sera from children allergic to milk. Isoelectric focusing was preferred to SDS-PAGE separation since it concentrates the protein bands (focusing effect) and is clearly much less

denaturing than SDS-PAGE. Moreover, it requires smaller volumes of sample, which represents an advantage when dealing with extremely limited serum volumes delivered by newborn patients or very young children. Figure 5 shows data from a first screening study of sera from 25 milk allergic patients against the initial milk proteins (lane “l”). Seven of them (the most active) were then selected to search for minor allergens: “e”, “f”, “g”, “k”, “m”, “o” and “p”. The selection was based on the specificity of IgE antibodies against known allergens. Selected sera were then used for the detection of allergens from library eluates (see Figure 6). While milk whey did not show visible immunoreaction for Ig (Figure 6A), eluates from the peptide libraries (especially Library-2, Figure 6B) revealed quite clearly the presence of Ig’s as allergens for examined sera of patients. Actually, the patient sera used here was “p” from Figure 5, well-known to comprise IgE against bovine Ig. In this IEF position, no other proteins are present, not even lactoferrin that normally shows a more alkaline isoelectric point. The sample that gave the highest Ig signal was the Library-2 eluate as also revealed by Coomassie staining (lane Lib-2 of Figure 6B); Library-1 gave a very low signal while the initial milk did not show significant immunodetectable reaction (Figure 6A). So far, an Ig allergic reaction was only reported for Ig heavy chain and with a very faint positive signal by Natale et al.12 after separation by two-dimensional electrophoresis. Beside polymorphic immunoglobulins, lactoferrin was Journal of Proteome Research • Vol. 8, No. 8, 2009 3929

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Table 1. List of Proteins Found in the Present Study after Treatment with Library-1 (Lib-1) and with Library-2 (Lib-2)a identified proteins

accession number

molecular mass

origin

LTF Lactotransferrin LGB Beta-lactoglobulin XDH 147 kDa protein ACTG1 Actin, cytoplasmic 2 IGHM IGHM protein B4GALT1 Isoform Long of Beta-1,4-galactosyltransferase 1 MFGE8 Isoform Long of Lactadherin ALB Serum albumin LOC780933;LOC615026 Cationic trypsin C3 Complement C3 (Fragment) PIGR Isoform Long of Polymeric immunoglobulin receptor LPO Lactoperoxidase GLYCAM1 Glycosylation-dependent cell adhesion molecule 1 - IGHG1 protein CLU Clusterin BTN1A1 Butyrophilin subfamily 1 member A1 Vl1a Vl1a protein LOC281812;LOC783341 Fibroblast growth factor-binding protein 1 IGLL1 24 kDa protein APOE Apolipoprotein E FGA Fibrinogen alpha chain CD14 CD14 protein IDH1 Isocitrate dehydrogenase [NADP] cytoplasmic GP2 Glycoprotein 2 APOA4 Apolipoprotein A-IV CATHL1 18 kDa protein LPL Lipoprotein lipase GC Vitamin D-binding protein LCN2 similar to lipocalin FGB Fibrinogen beta chain CHI3L1 chitinase 3-like 1 FGG 50 kDa protein FABP3 Fatty acid-binding protein, heart - Putative uncharacterized protein APOA1 Apolipoprotein A-I SERPING1 Factor XIIa inhibitor ENO1 Alpha-enolase GGT1 similar to Gamma-glutamyltranspeptidase 1 precursor IGL@ IGL@ protein LALBA Alpha-lactalbumin NUCB1 Nucleobindin-1 GSN gelsolin a TF Serotransferrin LOC789490 similar to Ig lambda chain V-I region BL2 NP Purine nucleoside phosphorylase LDHB L-lactate dehydrogenase B chain CATHL2 Cathelicidin-2 AZGP1 Zinc-alpha-2-glycoprotein CSN3 Kappa-casein GDI2 Rab GDP dissociation inhibitor beta GANAB glucosidase, alpha; neutral AB CSN1S1 Alpha-S1-casein FN1 fibronectin 1 isoform 12 SERPINA1 Alpha-1-antiproteinase ECHDC1 Enoyl-CoA hydratase domain-containing protein 1 CTSB Cathepsin B SCGB1D2 Secretoglobin, family 1D, member 2 ABCG2 ATP-binding cassette, subfamily G (WHITE), member 2 TKT Transketolase - 135 kDa protein A2M A2M protein CRISP3 Cysteine-rich secretory protein 3 LBP Lipopolysaccharide-binding protein ATP6AP2 ATP6AP2 protein SERPIND1 SERPIND1 protein CD36 Platelet glycoprotein 4

IPI00710664 IPI00699698 IPI00843038 IPI00712838 IPI00718725 IPI00685910 (+1) IPI00689035 IPI00708398 IPI00706427 (+1) IPI00713505 IPI00696714 IPI00716157 IPI00716366 IPI00855694 (+1) IPI00694304 IPI00708535 IPI00693917 IPI00704023 IPI00838162 IPI00712693 IPI00691819 IPI00686931 IPI00702781 IPI00695142 IPI00695965 IPI00717085 (+1) IPI00692291 IPI00823795 IPI00685784 IPI00709763 IPI00717764 IPI00843209 IPI00691946 IPI00852509 IPI00715548 IPI00710025 IPI00707095 IPI00705565 IPI00699011 IPI00717424 IPI00722271 IPI00883474 IPI00690534 IPI00906980 IPI00717573 (+1) IPI00760524 IPI00691669 IPI00698993 IPI00707811 IPI00713760 IPI00703243 IPI00706094 IPI00728194 (+2) IPI00695489 (+1) IPI00704943 IPI00692061 IPI00824879 (+2) IPI00690408 IPI00904104 IPI00717284 IPI00871133 IPI00715999 IPI00730056 IPI00693723 IPI00688367 IPI00710204

78 kDa 20 kDa 147 kDa 42 kDa 66 kDa 45 kDa 47 kDa 70 kDa 26 kDa 187 kDa 82 kDa 81 kDa 17 kDa 51 kDa 51 kDa 59 kDa 25 kDa 26 kDa 24 kDa 36 kDa 67 kDa 40 kDa 47 kDa 59 kDa 43 kDa 18 kDa 53 kDa 53 kDa 23 kDa 53 kDa 44 kDa 50 kDa 15 kDa 52 kDa 30 kDa 52 kDa 47 kDa 61 kDa 25 kDa 16 kDa 55 kDa 86 kDa 78 kDa 14 kDa 32 kDa 37 kDa 20 kDa 34 kDa 21 kDa 50 kDa 109 kDa 25 kDa 260 kDa 46 kDa 34 kDa 37 kDa 11 kDa 73 kDa 65 kDa 135 kDa 168 kDa 27 kDa 54 kDa 39 kDa 55 kDa 53 kDa

Ctrl + Lib-1 + Lib-2 Ctrl + Lib-1 + Lib-2 Ctrl + Lib-1 + Lib-2 Ctrl + Lib-1 + Lib-2 Ctrl + Lib-1 + Lib-2 Ctrl + Lib-1 + Lib-2 Ctrl + Lib-1 + Lib-2 Ctrl + Lib-1 + Lib-2 Ctrl + Lib-1 + Lib-2 Ctrl + Lib-1 + Lib-2 Ctrl + Lib-1 + Lib-2 Ctrl + Lib-1 + Lib-2 Ctrl + Lib-1 + Lib-2 Ctrl + Lib-1 + Lib-2 Ctrl + Lib-1 + Lib-2 Ctrl + Lib-1 + Lib-2 Ctrl + Lib-1 + Lib-2 Ctrl + Lib-1 + Lib-2 Ctrl + Lib-1 + Lib-2 Ctrl + Lib-1 + Lib-2 Ctrl + Lib-1 + Lib-2 Ctrl + Lib-1 + Lib-2 Ctrl + Lib-1 + Lib-2 Ctrl + Lib-1 + Lib-2 Ctrl + Lib-1 + Lib-2 Ctrl + Lib-1 + Lib-2 Lib-1 + Lib-2 Ctrl + Lib-1 + Lib-2 Ctrl + Lib-1 + Lib-2 Ctrl + Lib-1 + Lib-2 Ctrl + Lib-1 + Lib-2 Ctrl + Lib-1 + Lib-2 Ctrl + Lib-1 + Lib-2 Ctrl + Lib-1 + Lib-2 Ctrl + Lib-1 + Lib-2 Ctrl + Lib-2 Lib-1 + Lib-2 Ctrl + Lib-1 + Lib-2 Lib-1 Ctrl + Lib-1 + Lib-2 Ctrl Ctrl + Lib-2 Ctrl + Lib-2 Lib-1 + Lib-2 Lib-1 + Lib-2 Lib-1 Lib-1 + Lib-2 Ctrl + Lib-2 Lib-1 + Lib-2 Ctrl + Lib-1 + Lib-2 Lib-1 Ctrl + Lib-1 + Lib-2 Ctrl + Lib-1 Ctrl Lib-1 + Lib-2 Lib-1 Lib-1 Ctrl + Lib-2 Lib-1 + Lib-2 Lib-1 + Lib-2 Ctrl + Lib-1 Ctrl + Lib-1 + Lib-2 Lib-1 Lib-1 + Lib-2 Lib-1 + Lib-2 Ctrl + Lib-1 + Lib-2

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Exploration of Cow’s Whey Proteome Table 1. Continued identified proteins

accession number

molecular mass

origin

PDXK Pyridoxal kinase B2M Beta-2-microglobulin C9 Complement component C9 EZR Ezrin YKT6 Synaptobrevin homologue YKT6 LOC784964 similar to Endopin 1b isoform 1 GALM Aldose 1-epimerase LOC515150 similar to C4b-binding protein alpha chain precursor - 171 kDa protein MBL2 Mannose-binding protein C MUC15 Isoform 1 of Mucin-15 - 57 kDa protein FBP1 Folate receptor alpha ECM1 ECM1 protein HSPA8 Heat shock cognate 71 kDa protein LOC781223 similar to Hist1h4c protein TPP1 Tripeptidyl-peptidase 1 SIL1 Nucleotide exchange factor SIL1 ITIH4 Inter-alpha-trypsin inhibitor heavy chain H4 TNFRSF6B TNFRSF6B protein NELL2 NELL2 protein A1BG Alpha-1B-glycoprotein RNASE4 Ribonuclease, RNase A family, 4 M6PRBP1 Mannose-6-phosphate receptor binding protein 1 SLC34A2 Sodium-dependent phosphate transport protein 2B PPIA Peptidyl-prolyl cis-trans isomerase A RAB11A Ras-related protein Rab-11A NUCB2 Nucleobindin 2 LAP3 Isoform 1 of Cytosol aminopeptidase SDF4 Stromal cell derived factor 4 RAB7A Ras-related protein Rab-7a SCGB2A2 SCGB2A2 protein C4BPA C4b-binding protein alpha chain SERPINC1 Antithrombin-III PGLYRP1 Peptidoglycan recognition protein UGP2 UTP-glucose-1-phosphate uridylyltransferase LOC524176 similar to histone cluster 1, H2ag CP CP protein FBP1 Fructose-1,6-bisphosphatase 1 CFB Complement factor B (Fragment) HIST1H2BI Histone 1, H2bi ANG Angiogenin-1 LOC518318 similar to histone cluster 1, H3f MUC1 Mucin-1 RAB1A RAB1A, member RAS oncogene family LOC509924;LOC788112 LOC788112 protein XDH Xanthine dehydrogenase/oxidase CREG1 Cellular repressor of E1A-stimulated genes 1 AGP Alpha-1-acid glycoprotein PSAP Isoform 1 of Proactivator polypeptide CTSD Cathepsin D CFH Complement factor H TTR Transthyretin LRG1 Leucine-rich alpha-2-glycoprotein 1 MGC151921 Odorant-binding protein-like RAP1B Ras-related protein Rap-1b PSMB1 Proteasome subunit beta type-1 CD5L CD5L protein CPB2 Carboxypeptidase B2 HP Haptoglobin STCH Heat shock 70 kDa protein 13 KNG1 Isoform HMW of Kininogen-2 OS9 Protein OS-9 ENPP3 Ectonucleotide pyrophosphatase/ phosphodiesterase family member 3 C4A complement component 4A

IPI00701044 IPI00686769 IPI00715188 IPI00694641 IPI00708611 (+1) IPI00824495 IPI00712164 IPI00690298 IPI00687757 (+1) IPI00714518 IPI00716220 (+1) IPI00708969 IPI00708447 IPI00692534 (+1) IPI00708526 IPI00691248 (+5) IPI00721428 IPI00703448 IPI00717930 IPI00708920 IPI00867415 IPI00692686 IPI00760446 (+1) IPI00700098 IPI00703813 IPI00697285 (+1) IPI00695221 (+1) IPI00696729 IPI00702157 (+1) IPI00711047 IPI00704752 IPI00711254 IPI00702590 IPI00688316 IPI00701640 IPI00689863 (+1) IPI00689821 (+13) IPI00703491 IPI00704584 IPI00717527 (+1) IPI00701086 (+9) IPI00710136 (+1) IPI00701325 (+9) IPI00706283 IPI00829520 (+1) IPI00868525 IPI00695367 IPI00702458 IPI00691212 IPI00718311 (+1) IPI00715947 (+1) IPI00713757 IPI00689362 IPI00702820 IPI00730488 IPI00695776 IPI00722440 IPI00867131 IPI00685411 IPI00705491 IPI00685695 IPI00714019 (+1) IPI00706896 IPI00712650

35 kDa 14 kDa 62 kDa 69 kDa 22 kDa 46 kDa 38 kDa 22 kDa 171 kDa 26 kDa 36 kDa 57 kDa 28 kDa 58 kDa 71 kDa 11 kDa 61 kDa 53 kDa 102 kDa 32 kDa 91 kDa 54 kDa 17 kDa 48 kDa 76 kDa 18 kDa 24 kDa 49 kDa 56 kDa 41 kDa 24 kDa 10 kDa 69 kDa 52 kDa 21 kDa 57 kDa 14 kDa 121 kDa 37 kDa 85 kDa 14 kDa 17 kDa 15 kDa 58 kDa 23 kDa 20 kDa 147 kDa 24 kDa 23 kDa 58 kDa 42 kDa 140 kDa 16 kDa 38 kDa 20 kDa 21 kDa 26 kDa 50 kDa 49 kDa 45 kDa 52 kDa 69 kDa 76 kDa 100 kDa

Lib-1 Ctrl + Lib-1 + Lib-2 Lib-1 + Lib-2 Ctrl + Lib-1 + Lib-2 Lib-1 + Lib-2 Lib-2 Lib-1 Lib-2 Lib-1 + Lib-2 Lib-2 Ctrl + Lib-2 Lib-2 Ctrl + Lib-2 Lib-2 Lib-1 + Lib-2 Lib-1 Lib-1 Ctrl + Lib-1 + Lib-2 Ctrl + Lib-1 Lib-1 + Lib-2 Lib-2 Ctrl Lib-1 + Lib-2 Lib-2 Ctrl Lib-2 Lib-1 Ctrl Lib-1 + Lib-2 Ctrl Lib-1 Lib-1 Lib-2 Ctrl Lib-2 Lib-2 Lib-1 Ctrl Lib-1 Ctrl Lib-1 Lib-2 Lib-1 Ctrl + Lib-2 Ctrl + Lib-1 Lib-2 Lib-1 Ctrl Ctrl Lib-1 Lib-1 Lib-2 Ctrl Ctrl Ctrl Lib-1 + Lib-2 Lib-1 + Lib-2 Ctrl + Lib-2 Lib-2 Ctrl Lib-1 Ctrl Ctrl Ctrl

IPI00694498 (+1)

193 kDa

Ctrl

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Table 1. Continued identified proteins

SYNE1 Synaptic nuclear envelope protein MASP1 Mannan-binding lectin serine peptidase 1 MAN2A2 similar to mannosidase, alpha, class 2A, member 2 isoform 1 C7 93 kDa protein IGK IGK protein FUCA1 Tissue alpha-L-fucosidase HSPA1B Heat shock 70 kDa protein 1B CTSZ Cathepsin Z ACO1 Cytoplasmic aconitate hydratase PGAM1 Phosphoglycerate mutase 1 ANXA5 Annexin A5 SERPINA3-7 47 kDa protein F13B Coagulation factor XIII, B polypeptide F2 Prothrombin (Fragment) - 194 kDa protein ITIH1 Inter-alpha-trypsin inhibitor heavy chain H1 RBP4 retinol binding protein 4, plasma SPP1 Osteopontin a

molecular mass

origin

IPI00692470 IPI00708649 IPI00705260 IPI00703842 (+1) IPI00724838 (+2) IPI00732644 IPI00700035 (+1) IPI00708474 IPI00685374 IPI00698589 IPI00692093 (+1) IPI00686980 (+2) IPI00721056 IPI00710799 IPI00717116 IPI00686012 IPI00697184 IPI00691887 (+2)

1005 kDa 81 kDa 130 kDa 93 kDa 27 kDa 54 kDa 70 kDa 34 kDa 98 kDa 29 kDa 36 kDa 47 kDa 75 kDa 71 kDa 194 kDa 101 kDa 23 kDa 31 kDa

Lib-2 Lib-2 Lib-1 + Lib-2 Lib-2 Ctrl Lib-1 Lib-2 Ctrl Lib-1 Lib-1 Ctrl Ctrl Lib-2 Ctrl Lib-2 Ctrl Ctrl Ctrl

Crtl is the initial nontreated milk whey proteome.

Figure 3. (A) Overlapping Venn diagrams of the proteins detected in the initial milk whey material (Ctrl) and two eluates from Library-1 and Library-2. (B) Comparison of gene product found by merging all data from the present study and largest reported protein list from whey investigation.12

also detectable at pI 9.5 (see, for example, Figure 6B track “k”). Many other minor nonidentified allergens were clearly identified by all sera except “g”, from library eluates in the very acidic part of the gel, between pI 3 and 4. These data are preliminary and they necessarily deserve more in depth investigations. However, it was important to report this early interesting discovery, enlarging thus the possibility to focus on the aspect of this newly developed method capable of revealing lowabundance species.

Discussion and Conclusion At least two major findings emerge from the present study: (i) a number of novel proteins was found in the whey proteome after treatment with the peptide libraries; (ii) allergens were much more visible and for the first time polymorphic Ig’s were detected as allergenic antigens. As a corollary of the above, it seems that we will have to live with the notion that, even in simple proteomes, their complexity is much larger than expected. Some instructive examples are given below. Up to the present, it was believed that egg white would be constituted 3932

accession number


by a handful of major proteins. In fact, a recent article,27 exploiting most powerful mass spectrometers (FT-ICR), all at once demonstrated that 78 unique protein species were present. Although this was thought to be an incredible number, when we applied to this system the CPLL technology, this figure was almost doubled (via CPLL treatment) to 148 unique gene products,28 although much less sophisticated MS instrumentation was used. Similar results were obtained when exploring chicken egg yolk. Here too, the proteomic landscape was all at once increased to a grand total of 100 species by Mann and Mann;29 yet, when we applied the CPLLs, no less than 255 unique gene products could be counted,30 an incredible number, considering that here too only a handful of proteins are displayed in SDS-PAGE. Even at the onset of the CPLL technique, when we explored the human urinary proteome, whereas in the control, untreated sample, only 96 unique species could be identified, after CPLL treatment, a total of 497 unique proteins could be displayed, covering a very large mass interval, from 10 up to 220 kDa.31 But perhaps the most striking results were obtained when probing the cytoplasmic proteome of human red blood cells (RBC). It is known that RBCs contain almost 98% hemoglobin, the remainder 2% minority proteome being largely unknown. Haematologists, over decades of work, had reported perhaps up to 50-60 enzymes in the RBC cytoplasm, mostly connected with maintenance of glycolysis, cation pumping against electrochemical gradients, synthesis of glutathione and other metabolites, nucleotide catabolism reactions, maintenance of hemoglobin iron in its functional ferrous state and protection of enzymatic and structural proteins from oxidative denaturation. Here too, with modern proteomic techniques, a total of 252 species were identified by Pasini et al.32 Although this number is outstanding, it still falls short of expectations, considering that the cytoplasmic proteomic asset would be inherited by the erythroblasts and that it is believed that living human cells should have a genetic asset of >10 000 unique gene products fully expressed. When applying to this same system the CPLL methodology, a grand total of 1570 unique gene products was discovered,33 a 5× increment over the best report ever published! How is all this possible? One explanation for that is that we work with the CPLLs under very large overloading conditions,

research articles

Exploration of Cow’s Whey Proteome

Figure 4. Gene Ontology (GO) functional classification of proteins from initial cow whey (A) and after treatment with peptide ligand libraries (B) merged lists, after having removed redundancies.

Table 2. List of Exclusive Proteins Found in the Eluates of Library-1 and Library-2 after Withdrawal of Proteins Common to Literature12 Library-1 accession number

Library-2

identified protein

accession number

identified protein

IPI00824495 IPI00714518 IPI00708969 IPI00692534 (+1) IPI00867415

LOC784964 similar to Endopin 1b isoform 1 MBL2 Mannose-binding protein C - 57 kDa protein ECM1 ECM1 protein NELL2 NELL2 protein

IPI00700098

IPI00701044

IGL@ IGL@ protein LDHB L-lactate dehydrogenase B chain GANAB glucosidase, alpha; neutral AB CTSB Cathepsin B SCGB1D2 Secretoglobin, family 1D, member 2 LBP Lipopolysaccharide-binding protein PDXK Pyridoxal kinase

IPI00712164

GALM Aldose 1-epimerase

IPI00689863 (+1)

IPI00691248 (+5)

IPI00710136 (+1)

IPI00721428

LOC781223 similar to Hist1h4c protein TPP1 Tripeptidyl-peptidase 1

M6PRBP1 Mannose-6-phosphate receptor binding protein 1 PGLYRP1 Peptidoglycan recognition protein UGP2 UTP-glucose-1-phosphate uridylyltransferase ANG Angiogenin-1

IPI00695221 (+1) IPI00704752 IPI00711254

RAB11A Ras-related protein Rab-11A RAB7A Ras-related protein Rab-7a SCGB2A2 SCGB2A2 protein

IPI00713757 IPI00685411 IPI00692470

IPI00689821 (+13)

LOC524176 similar to histone cluster 1, H2ag FBP1 Fructose-1,6-bisphosphatase 1 HIST1H2BI Histone 1, H2bi LOC518318 similar to histone cluster 1, H3f STCH Heat shock 70 kDa protein 13 FUCA1 Tissue alpha-L-fucosidase ACO1 Cytoplasmic aconitate hydratase PGAM1 Phosphoglycerate mutase 1

IPI00708649

IPI00699011 IPI00760524 IPI00703243 IPI00692061 IPI00824879 (+2) IPI00730056

IPI00704584 IPI00701086 (+9) IPI00701325 (+9) IPI00685695 IPI00732644 IPI00685374 IPI00698589

IPI00701640

IPI00868525

IPI00703842 (+1) IPI00700035 (+1) IPI00721056 IPI00717116

LOC509924;LOC788112 LOC788112 protein CFH Complement factor H CPB2 Carboxypeptidase B2 SYNE1 Synaptic nuclear envelope protein MASP1 Mannan-binding lectin serine peptidase 1 C7 93 kDa protein HSPA1B Heat shock 70 kDa protein 1B F13B Coagulation factor XIII, B polypeptide - 194 kDa protein


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D’Amato et al.

Figure 5. Immunoblotting from an IEF separation of whole fresh milk serum against sera from 25 allergic patients (lanes “a” through “z”), and a negative control (lane “I”). Sample applied as a linear deposit on a line parallel to the electrodes and at 1 cm from the anode. The major allergens recognized are caseins (cas), β-lactoglobulin (βLg) and R-lactalbumin (RLa), both at the same pI. Positioning of minor allergens is indicated for lactoferrin (Lf) and the bovine Immunoglobulins (Ig).

Figure 6. (A) Immunoblotting from an IEF analysis of initial milk whey proteins against limited sera samples. The “wp” lane is the crude whey protein mixture stained with Coomassie blue. The following lanes on the right are immunoblots of the same protein mixture with sera “e”, “o”, “m”, “k”, “g”, “f” and “p”. “ct” is the negative control. (B) Immunoblotting from an IEF analysis of library eluates against the same sera samples listed in panel A. Lanes “Lib-1” and “Lib-2” are Coomassie stained eluates, while all others are as described for panel A, all of them from Library-2. For both panels A and B, regions indicated by “cas”, “R+β”, “Ig” and “Lf” mean, respectively, where R-lactalbumin, β-lactoglobulin, immunoglobulins and lactoferrin are generally detected.

that is, under an experimental setup that defies general chromatographic rules, which require that columns be operated well below the saturation level of the total binding sites on the resin. For proper results and maximum separation efficiency, in fact, chromatographic columns should be loaded with protein levels that would saturate at maximum 20% of the binding sites; when developing the column, thus, on the way to the outlet, the separands would continuously bind and be displaced from the large majority of binding sites still free, so as to maximize separation. Conversely, with the CPLLs, the large overloading conditions, well above the saturation level for the high-to-medium-abundance proteins, allow proteins present in traces to be continuously adsorbed onto their 3934


respective ligand, not only via direct affinity to them, but also via the law of mass action that would shift the equilibrium toward to bound state. It is thus quite probable that most (if not all) of the low- to very-low-abundance species will be sequestered onto the resin, resulting in a strong amplification of the signal. Such amplification can be estimated by observing that, for example, in the present case, we have loaded as much as 500 mL of whey. Considering that in most biological investigation often as little as 100 µL of sample is utilized (but in capillary zone electrophoresis only nanoliter volumes are injected), if the species present in traces had been almost quantitatively recovered onto the beads, this would mean an “amplification” of the signal by at least 3 orders of magnitude.

research articles

Exploration of Cow’s Whey Proteome And this should be operative for most of the species present in the sample, considering that CPLLs can be envisioned as an affinity column, but endowed with millions of different affinity baits, represented by the hexapeptides present on the beads. To our knowledge, there is only another system that can be operative at the same level, namely, disc electrophoresis, as originally envisioned by Ornstein34 and Davis.35 When a proteinaceous sample is properly inserted between a leading and a terminating ion, and an electric field is applied, a number of discontinuities in the system permit a stacking effect that can easily reach a concentration of the sample species by 3 orders of magnitude. This is acting as long as the isotachophoretic train is operative; however, as the system is changed from isotachophoretic conditions to conventional zone electrophoresis, band diffusion ensues and resolution and concentration are lost as the electrophoretic run progresses. With the capabilities of CPLLs, it was thus possible to identify a quite large number of new gene products that, if added to the most comprehensive list,12 would engender a total of 257 species. This number could eventually be enlarged with the investigation of colostrum proteome, the latter containing more immunoglobulins and presumably additional associated lowabundance species. Interestingly the contribution in new species from the present list (see Table 2) is essentially related to low-abundance species that could not be detected previously and could possibly comprise among other proteins unknown allergens. In fact, in addition to classical major allergens so far reported in milk, such as caseins, R-lactalbumin and β-lactoglobulin, some minor ones are known as BSA, bovine immunoglobulins chains and lactoferrin. Most frequently, the signal of these allergens is covered by the presence of major proteins as they have either a common pI range or a close mass. Even in ELISA tests, when too high IgE antibody titers are binding to the major allergens, the detection of the minor ones can be inhibited, maybe by steric hindrance, as small amounts of IgE will not be able to bind to these minor allergens. Our great surprise was to see that all 7 patient sera used were capable to recognize polymorphic bovine Ig’s as allergens (Figure 6B) even those which showed negative reaction with Ig in Figure 6A such as serum “e”, serum “m” and serum “g”. These detections were only possible because of the amplification of the signal of these low-abundance allergens, concomitant with the decrease of the concentration of high-abundance allergens. Bovine Ig’s were located in the alkaline part of the IEF and were also consistent with the detection of single multiple heavy and light chains spots in two-dimensional electrophoresis for the eluate from Library-2, all of them located in the alkaline region of the plate, but almost absent from the eluate of Library-1. Bovine serum albumin was not easily detected by immunoblot as it was hidden among the remaining and very heterogeneous casein bands. A strong increase in the detectability of lactoferrin as an allergen was also observed. As indicated in Results, very acidic allergens, so far unknown, were also detected with several allergic patient sera. They will be identified by peptide sequencing using mass spectrometry. This heavy, but important, pioneering work is in progress in collaboration with other laboratories. In conclusion, this first use of peptide library treatment of milk proteins allowed evidencing new species not yet known as milk proteins; additionally, among the proteins found, it was possible to identify minor allergens that generally are covered by the signal of immunological reactions generated by major

allergenic antigens. After the detection of low-abundance allergens from corn seed extracts,36 it is confirmed that the amplification of low-abundance species (as well engendered by combinatorial peptide libraries) is a way to detect allergens of very low concentration. Although still very preliminary, these finding should stimulate novel discovery activity in order to confirm that allergens could be found from species that are not detectable from untreated biological samples.

Acknowledgment. P.G.R. was supported by Fondazione Cariplo (Milano), by PRIN 2009 (MURST, Rome) and by an ERC (European research Council) Senior Investigator Grant. A.B. is partially supported by Fondazione Cariplo (Progetto Nobel: Guard). References (1) Swaisgood, H. E. In Handbook of Milk Composition; Jensen, R. G., Ed.; Academic Press: New York, 1995; pp 464-467. (2) Anderson, N. G.; Powers, T.; Tollaksen, S. L. Proteins of human milk. I. Identification of major components. Clin. Chem 1982, 28, 1045–1055. (3) Galvani, M.; Hamdan, M.; Righetti, P. G. Two-dimensional gel electrophoresis/matrix assisted laser desorption/ionization mass spectrometry of a milk powder. Rapid Commun. Mass Spectrom. 2000, 14, 1889–1897. (4) Galvani, M.; Hamdan, M.; Righetti, P. G. Two-dimensional gel electrophoresis/matrix assisted laser desorption/ionization mass spectrometry of commercial bovine milk. Rapid Commun. Mass Spectrom. 2001, 15, 258–264. (5) Yamada, M.; Murakami, K.; Wallingford, J. C.; Yuki, Y. Identification of low-abundance proteins of bovine colostral and mature milk using two-dimensional electrophoresis followed by microsequencing and mass spectrometry. Electrophoresis 2002, 23, 1153–1160. (6) Reinhardt, T. A.; Lippolis, J. D. Bovine milk fat globule membrane proteome. J. Dairy Res. 2006, 73, 406–416. (7) Smolenksi, G.; Haines, S.; Kwan, F. Y. S.; Bond, J.; et al. Characterisation of host defence proteins in milk using a proteomic approach. J. Proteome Res. 2007, 6, 207–215. (8) D’Ambrosio, C.; Arena, S.; Salzano, A. M.; Renzone, G.; et al. A proteomic characterization of water buffalo milk fractions describing PTM of major species and the identification of minor components involved in nutrient delivery and defense against pathogens. Proteomics 2008, 8, 3657–3666. (9) Cavaletto, M.; Giuffrida, M. G.; Conti, A. The proteomic approach to analysis of human milk fat globule membrane. Clin. Chim. Acta 2004, 347, 41–48. (10) Fortunato, D.; Giuffrida, M. G.; Cavaletto, M.; Garoffo, L. P.; et al. Structural proteome of human colostral fat globule membrane proteins. Proteomics 2003, 3, 897–905. (11) Conti, A.; Giuffrida, G.; Cavaletto, M. Proteomics of human milk. In Proteomics of Human Body Fluids: Principles, Methods and Applications; Thongboonkerd, V., Ed.; Human Press; Totowa, 2007; pp 437-451. (12) Palmer, D. J.; Kelly, V. C.; Smit, A. M.; Kuy, S.; et al. Human colostrum: identification of minor proteins in the aqueous phase by proteomics. Proteomics 2006, 6, 2208–2216. (13) Righetti, P. G.; Boschetti, E.; Lomas, L.; Citterio, A. Protein Equalizer technology: the quest for a “democratic proteome”. Proteomics 2006, 6, 3980–3992. (14) Righetti, P. G.; Boschetti, E. Sherlock Holmes and the proteomesa detective story. FEBS J. 2007, 274, 897–905. (15) Boschetti, E.; Lomas, L.; Citterio, A.; Righetti, P. G. Romancing the “hidden proteome”, Anno Domini two zero zero seven. J. Chromatogr., A 2007, 1153, 277–290. (16) Boschetti, E.; Monsarrat, B.; Righetti, P. G. The “invisible proteome”: how to capture the low abundance proteins via combinatorial ligand libraries. Curr. Proteomics 2007, 4, 198–208. (17) Boschetti, E.; Righetti, P. G. Hexapeptide combinatorial ligand libraries: the march for the detection of the low-abundance proteome continues. BioTechniques 2008, 44, 663–665. (18) Boschetti, E.; Righetti, P. G. The ProteoMiner in the proteomic arena: A non-depleting tool for discovering low-abundance species. J. Proteomics 2008, 71, 255–264. (19) Righetti, P. G.; Boschetti, E. The ProteoMiner and the FortyNiners: Searching for gold nuggets in the proteomic arena. Mass Spectrom. Rev. 2008, 27, 596–608.


research articles (20) Guerrier, L.; Righetti, P. G.; Boschetti, E. Reduction of dynamic protein concentration range of biological extracts for the discovery of low-abundance proteins by means of hexapeptide ligand library. Nat. Protoc. 2008, 3, 883–890. (21) Candiano, G.; Bruschi, M.; Musante, L.; Santucci, L.; et al. Blue Silver: a very sensitive colloidal Coomassie G-250 staining for proteome analysis. Electrophoresis 2004, 25, 1327–1333. (22) Hamdan, M.; Righetti, P. G. Proteomics Today; Wiley: Hoboken, 2005; pp 346-348. (23) Simo´, C.; Bachi, A; Cattaneo, A.; Guerrier, L.; Fortis, F; Boschetti, E.; Podtelejnikov, A.; Righetti, P. G. Performance of combinatorial peptide libraries in capturing the low-abundance proteome of red blood cells. 1. Behavior of mono- to hexapeptides. Anal. Chem. 2008, 80, 3547–3556. (24) Olsen, J. V.; de Godoy, L. M.; Li, G.; Macek, B.; et al. Parts per million mass accuracy on an Orbitrap mass spectrometer via lock mass injection into a C-trap. Mol. Cell. Proteomics 2005, 12, 2010– 2021. (25) Craig, R.; Beavis, R. C. TANDEM: matching proteins with tandem mass spectra. Bioinformatics 2004, 20, 1466–1467. (26) Natale, M.; Bisson, C.; Monti, G.; Peltran, A.; et al. Cow’s milk allergens identification by two-dimensional immunoblotting and mass spectrometry. Mol. Nutr. Food Res. 2004, 48, 363–369. (27) Mann, K. The chicken egg white proteome. Proteomics 2007, 7, 3558–3568.

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D’Amato et al. (28) D’Ambrosio, C.; Arena, S.; Scaloni, A.; Guerrier, L.; et al. Exploring the chicken egg white proteome with combinatorial peptide ligand libraries. J. Proteome Res. 2008, 7, 3461–3474. (29) Mann, K.; Mann, M. The chicken egg yolk plasma and granule proteomes. Proteomics 2008, 8, 178–191. (30) Farinazzo, A.; Restuccia, U.; Bachi, A.; Guerrier, L.; et al. Chicken egg yolk cytoplasmic proteome, mined via combinatorial peptide ligand libraries. J. Chromatogr., A 2009, 1216, 1241–1252. (31) Castagna, A.; Cecconi, D.; Sennels, L.; Rappsilber, J.; et al. “Exploring the hidden human urinary proteome via ligand library beads”. J. Proteome Res. 2005, 4, 1917–1930. (32) Pasini, E. M.; Kirkegaard, M.; Mortensen, P.; Lutz, H. U.; et al. Indepth analysis of the membrane and cytosolic proteome of red blood cells. Blood 2006, 108, 791–801. (33) Roux Dalvai, F.; Gonzalez de Peredo, A.; Simo´, C.; Guerrier, L.; et al. Extensive analysis of the cytoplasmic proteome of human erythrocytes using the peptide ligand library technology and advanced mass spectrometry. Mol. Cell. Proteomics 2008, 7, 2254– 2269. (34) Ornstein, L. Disc electrophoresis. I: background and theory. Ann. NY Acad. Sci. 1964, 121, 321–349. (35) Davis, B. J. Disc electrophoresis. II: method and application to human serum proteins. Ann. NY Acad. Sci. 1964, 121, 404–427. (36) Fasoli, E.; Pastorello, E. A.; Farioli, L.; Scibilia, J.; et al. Searching for allergens in maize kernels via proteomic tools. J. Proteomics 2009, 72, 501–510.

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In-Depth Exploration of Cow's Whey Proteome via Combinatorial

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