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Sample preparation of serum to allow capillary electrophoresis analysis of prostate specific antigen isoforms
Journal of Pharmaceutical and Biomedical Analysis 134 (2017) 220–227
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Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Sample preparation of serum to allow capillary electrophoresis analysis of prostate specific antigen isoforms Noemi Farina-Gomez a , Silvia Barrabes b , Jorge E. Gomez-Lopez a,c,1 , Monica Gonzalez a , Angel Puerta a , Diana Navarro-Calderon a , Eduardo Albers-Acosta d , Carlos Olivier d , Jose C. Diez-Masa a , Rosa Peracaula b,∗ , Mercedes de Frutos a,∗∗ a
Institute of Organic Chemistry (IQOG-CSIC), Madrid, Spain Unit of Biochemistry, Faculty of Sciences, University of Girona, Girona, Spain c Department of Chemistry, Universidad del Valle, Cali, Colombia d Department of Urology, Hospital La Princesa, Madrid, Spain b
a r t i c l e
i n f o
Article history: Received 28 July 2016 Received in revised form 21 November 2016 Accepted 23 November 2016 Available online 24 November 2016 Keywords: Sample treatment Serum Prostate specific antigen Capillary electrophoresis Two-dimensional electrophoresis Prostate cancer biomarker
a b s t r a c t Prostate cancer is the second most frequently diagnosed cancer in men worldwide. Currently prostate specific antigen (PSA) serum concentration is the most used prostate cancer marker, but it only shows limited specificity. Because PSA glycosylation is altered by prostate cancer, detecting glycosylation changes could increase PSA specificity as a prostate cancer marker. Changes in PSA glycosylation can modify its electrophoretic- behavior and techniques such as capillary zone electrophoresis (CZE) and two-dimensional electrophoresis (2-DE) could be applied to detect changes in PSA glycosylation. Most serum PSA is complexed with alpha-1 antichymotrypsin (ACT). To have access to most of the PSA, the complexed PSA has to be released as free PSA (fPSA); in addition, this total fPSA must be purified from the serum matrix so that it can be analyzed using CZE. In this work a methodology for isolating PSA from serum for its CZE analysis was established. By using PSA standard, the effect of this methodology, which combines conditions for dissociating complexed PSA and immunoaffinity chromatographic purification, was studied. It was seen that this highly repeatable sample treatment did not noticeably alter the circular dichroism (CD) spectrum or the CZE pattern of PSA standard. Therefore, as a proof-of-concept, the developed sample treatment was applied to serum from a cancer patient with a high PSA content. The following observations can be made from these experiments: first of all, the 2-DE pattern of serum PSA remained unchanged after sample treatment; second, as hypothesized, the established sample preparation methodology made it possible to obtain the CZE pattern of PSA from serum; and third, the CZE pattern of serum PSA and of PSA standard from seminal plasma of healthy individuals, both submitted to the sample treatment method, showed some differences regarding the proportion of CZE peaks of the glycoprotein. These differences could be related to possible changes in the linkages of peptide backbone, in glycosylation or in other post-translational modifications between samples from both origins. © 2016 Elsevier B.V. All rights reserved.
1. Introduction
Abbreviations: ACT, alpha-1 antichymotrypsin; 2-DE, two-dimensional electrophoresis; fPSA, free PSA; HRP, horseradish peroxidase; IAC, immunoaffinity chromatography; PSA, prostate specific antigen; tPSA, total PSA; Tris, Tris (hydroxymethyl) aminomethane; WB, Western blot. ∗ Corresponding author at: Biology Department, Faculty of Sciences, University of Girona, 17071, Girona, Spain. ∗∗ Corresponding author at: Department of Instrumental Analysis and Environmental Chemistry, Institute of Organic Chemistry, Spanish Research Council (IQOG- CSIC), Juan de la Cierva 3, 28006, Madrid, Spain. E-mail addresses: [email protected] (R. Peracaula), [email protected] (M. de Frutos). 1 Present address: Department of Chemistry, Universidad Nacional de Colombia, Bogotá, Colombia. http://dx.doi.org/10.1016/j.jpba.2016.11.045 0731-7085/© 2016 Elsevier B.V. All rights reserved.
In 2012, over 14 million new cancer cases and more than 8 million cancer deaths were reported worldwide. Out of them, 1.1 million corresponded to new cases of prostate cancer, which is the second most frequently diagnosed cancer in men worldwide [1]. While five-year survival for prostate cancer is estimated to be >99% for localized cancer, survival markedly decreases to less than 30% for distant cancer [2]. Controversial results on how cancer mortality is affected by screening based on serum prostate specific antigen (PSA) concentration are being published [3]. Its main drawback is its limited specificity, since non-malignant prostate diseases can also increase
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the level of serum PSA. Thus, the need for improved prostate cancer diagnostic methods has been emphasized [4]. In addition to genomic, proteomic, and metabolomic advances in the search for new biomarkers, approaches based on glycomics and glycoproteomics are also being applied because changes in glycosylation have been associated with cancer development and progression [5,6]. Specifically, modifications in the glycosylation of PSA and other proteins have been described in prostate cancer [7–10] and some of them, in particular core fucosylation levels and percentage of ␣2,3 sialic acid, have recently been described to identify aggressive prostate cancer and could, therefore, be useful to improve prostate cancer diagnosis [11]. Changes in glycosylation can alter the size and/or charge of the different molecular forms of a given glycoprotein and, therefore, their migration when analyzed by electrophoretic techniques such as two-dimensional electrophoresis (2-DE) or capillary electrophoresis (CE) can also be altered. 2-DE analyses of PSA have shown that the obtained PSA spot pattern can be partially explained by the different sialic acid content of PSA N-glycan chains [12]. However, techniques that are faster, more quantitative and easier to automate than 2-DE are desirable to reveal changes in the electrophoretic pattern of PSA. CE has these qualities and using this technique the patterns of some non-hydrolyzed glycoproteins have been shown to be potential disease biomarkers [13,14]. PSA is present in serum as a complex mixture of several species. The most abundant form is a covalent complex of PSA with alpha-1 antichymotrypsin (ACT) through an ester linkage. Free PSA (fPSA), including nicked forms corresponding to internally cleaved PSA polypeptide chains, accounts for only 5–30% of the total PSA (tPSA) [15]. In order to investigate if the CE pattern of serum PSA could be useful as a prostate cancer biomarker it is critical to use appropriate CE and sample preparation methods. The CE method should be able to separate PSA isoforms (peaks containing one or more glycoforms of fPSA). Such a CE method has been recently optimized by our group [16]. The sample preparation method should provide a high purity of the glycoprotein from the biological fluid without altering its CE profile. However, a serum treatment that makes it possible to purify PSA from it in a way that is compatible with CE analysis is not available. An immunoaffinity chromatography (IAC) method has been developed for isolating PSA from seminal plasma, where it exists as fPSA at high concentration [17]. However, most of PSA in serum exists as complexed PSA and its concentration is much lower than in seminal plasma. To have access to most of the tPSA, which can be more representative of modifications that take place due to prostate cancer [18], and to increase the available concentration of fPSA to be analyzed by CE, the ACT-PSA complex has to be dissociated. A method that employs ethanolamine to release PSA from this complex has already been described [15]. This method has been applied with some modifications to release PSA for 2-DE analysis under reducing conditions [18]. The CE methods developed to analyze fPSA isoforms work under non-reducing conditions [16,19,20]. Therefore, the five disulphide bridges of PSA held the protein structure together, including the nicked forms. This means that PSA isoform profile will be different if the analysis methods are performed under reducing conditions or under non-reducing ones. PSA released from serum by ethanolamine treatment was found to be very similar to PSA standard when using immunoassays, HPLC and MS [15]. However, the authors of this treatment indicated that it would be interesting to perform some experiments, including immunorecognition, regarding the structural integrity of PSA dissociated from the complex. The feasibility of purifying PSA from serum using an antiPSA affinity column, previously described for seminal plasma PSA
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purification, has not been addressed. In this regard, the effect of ethanolamine treatment on PSA recognition by the anti-PSA antibodies of the immunocolumn needs to be tested. In addition, the influence of a different matrix composition and of a much larger sample volume to compensate for the low PSA concentration in serum has not been studied. Furthermore, the effect of ethanolamine in combination with immunopurification on PSA structure and on its capillary electrophoresis pattern under nonreducing conditions has not been addressed. These questions, which must be resolved in order to validate the usefulness of the CE pattern of PSA as prostate cancer marker in the future, are studied in the present work. Specifically, a methodology to treat PSA with the dissociating agent and to purify the free glycoprotein using a house-made anti-PSA column that preserves separation of PSA isoforms by CE was developed using fPSA standard. SDS-PAGE followed by Western blot (WB), CD and CZE were used to control possible protein alterations during the different steps of the method. Finally, the usefulness of the sample preparation methodology for a clinical sample was tested by applying it to a prostate cancer patient’s serum and analyzing PSA from this fluid by CE and 2-DE.
2. Experimental 2.1. Samples, reagents, and devices 2.1.1. Materials ® The European standard (Certified Reference Material BCR 613) of fPSA purified from human seminal plasma used to develop the methodology was acquired from Isostandards Material (Madrid, Spain). The monoclonal (clone 5G6) anti-tPSA antibody from Antibodies on-line (Aachen, Germany) as well as the rest of materials used to fabricate the 3 cm x 2.1 mm I.D. anti-PSA column were as previously described [17]. Tris (hydroxymethyl) ® aminomethane (Tris), urea, Brij35 , propionic acid, sodium tetraborate, and decamethonium bromide were from Sigma-Aldrich (Steinheim, Germany). Sodium azide was from J.T. Baker (London, UK). Hydrochloric acid was from Panreac (Barcelona, Spain). Ethanolamine was from Fluka (Steinheim, Germany). Disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, and potassium chloride were from Merck (Darmstadt, Germany). The phosphate buffered saline (PBS) consisted of disodium hydrogen phosphate (0.1 M)/sodium dihydrogen phosphate (0.1 M) buffer with NaCl (1.38 M) and KCl (27 mM) (pH 7.4). ® Microcon YM-10 devices were from Millipore (Billerica, MA, USA). Fused silica capillaries were provided by CM Scientific Limited (Silsden, UK). The following reagents were used to perform SDS-PAGE, 2DE and Western blot (WB): 40% polyacrylamide solution and horseradish peroxidase (HRP) conjugated goat anti-rabbit IgG (H + L) were from Thermo Fisher Scientific (Waltham, MA, USA). Tris, Tris-HCl, glycine, SDS, ß-mercaptoethanol, bromophenol blue, urea and Triton X-100 were from Serva (Heidelberg, Germany). Glycerol and bovine serum albumin (BSA) were from Roche Diagnostics (Mannheim, Germany). PVDF membranes and Immobilon Western chemiluminescent HRP substrate were from Millipore. Methanol was from Panreac. Tween-20 was from Sigma-Aldrich. Dithiothreitol (DTT), iodoacetamide, pharmalyte 3–10 and Immobiline DryStrip gels (IPG strips) 18 cm pH 3–10 linear were from GE Healthcare (Buckinghamshire, UK). Agarose was from Ecogen (Barcelona, Spain) and polyclonal rabbit anti-human PSA was from Dako (Glostrup, Denmark). In all the experiments water was of milli-Q quality and it was obtained from a Millipore purification system (Bedford, MA).
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A serum sample from a prostate cancer patient with very high PSA concentration (>20 000 ng mL−1 ) was obtained at the Hospital de la Princesa (Madrid, Spain) following the guidelines of the hospital’s Ethical Committee. 2.1.2. Sample handling Lyophilized European PSA standard was dissolved in water (1 mg mL−1 ), aliquoted, and stored at −20 ◦ C until use. For SDSPAGE, it was diluted 1:9 (v/v) in water and 100 ng were loaded in Laemmli buffer (Tris/HCl (30 mM), SDS (2%, w/v), glycerol (10%, v/v), bromophenol blue (0.0125%, w/v), (pH 6.8)) with mercaptoethanol (1.25%, v/v); for IAC, it was diluted 1:1 (v/v) with 2× concentrated PBS. The serum sample was aliquoted and kept at −20 ◦ C until use. 2.2. Methods and equipment 2.2.1. Dissociation of PSA-ACT complex Treatment with ethanolamine was performed according to Peter et al. [15]. In short, 200 L PSA standard (30 g) or 175 L serum sample were treated with 2 M ethanolamine in PBS to get a 0.1 M final concentration of ethanolamine. The mixture was adjusted to pH 10.3 with HCl and incubated at 25 ◦ C for 24 h. Before PSA isolation, the reaction was stopped by removing ethanolamine using centrifugal filter devices with a 10 kDa cut-off membrane (Micro® con YM-10). 2.2.2. Polyacrylamide gel electrophoresis (1-DE and 2-DE) and western blot to immunodetect PSA SDS-PAGE was performed using a 12% polyacrilamide gel in resolving buffer (Tris-HCl (pH 8.8, 1.5 M), SDS (0.4%)) and a 5% polyacrilamide gel in stacking buffer (Tris-HCl (pH 6.8, 0.5 M), SDS (0.4%)). Samples were prepared with Laemmli buffer. Gel was run for 2 h at 100 V in running buffer (glycine (192 mM), Tris-HCl (25 mM), (pH 8.4), SDS (0.1%)), and transferred to a PVDF membrane for 3 h at 100 V in Towbin buffer (Tris (25 mM), glycine (192 mM), (pH 8.3), methanol (20%)). The membrane was blocked with a 3% BSA solution in Tris buffered saline-Tween (TBST) (Tris (10 mM), NaCl (100 mM), Tween-20 (0.1%)). PSA was detected using a polyclonal rabbit anti-PSA antibody diluted 1/4000 in 0.5% BSA in TBST, followed by a secondary goat anti-rabbit horseradish peroxidaseconjugated antibody diluted 1/80000 in 0.5% BSA in TBST. Several washing steps with TBST were performed between each step. Detection was performed using the Immobilon Western Chemiluminescent HRP Substrate and chemiluminescence was visualized using the Fluorochem SP imaging system (Alpha Innotech, San Leandro, CA, USA) under non-saturating conditions. Densitometry analysis was performed with AlphaEase FC Imaging system software (Alpha Innotech). 2-DE was performed as described previously [18] with minor modifications: In brief, samples (serum and PSA purified from the same serum) were diluted in 360 L of rehydration buffer (urea (8 M), Triton X-100 (0.5%), DTT (13 mM), Pharmalyte 3–10 (1%)) and were loaded in IPG strips pH 3–10, 18 cm by active in-gel rehydration. Isoelectric focusing was performed for a total run of 47 kV h. After the isoelectric focusing, the strips were cut to 7 cm keeping just the 5.5–7.5 pH range. For the second dimension the strips were first equilibrated for 15 min in equilibration buffer (urea (6 M), glycerol (30%, v/v), SDS (2%), Tris (50 mM), (pH 8.8)) containing 65 mM DTT and afterwards for 15 min in equilibration buffer containing 135 mM iodoacetamide. The strips were then placed on top of 10% polyacrylamide gels and sealed with a 0.5% agarose solution in running buffer (glycine (192 mM), Tris-HCl (25 mM), (pH 8.3–8.5), SDS (0.1%)) and traces of bromophenol blue. The second dimension was performed in a Miniprotean III unit (Bio-Rad, Hercules, CA, USA) with the running buffer described above and run at 1 W per gel.
Proteins were then transferred on a PVDF membrane and Western blot was carried out as described above.
2.2.3. Immunoaffinity isolation The method used was slightly modified from the one previously developed [17]. Briefly, PBS was pumped through the IAC column at 0.5 mL min−1 for 20 min before sample injection. Then, PSA standard was injected into the IAC column. PBS (0.5 mL min−1 ) was used as mobile phase. When baseline was recovered, 1 mL of 1 M propionic acid was injected into the column in order to elute the retained PSA and to collect it. Elution with propionic acid was carried out in triplicate, and the eluted fractions were collected on 1 M Tris. This method was modified throughout the present work, and the addition of 1 M Tris to the fraction eluted with propionic acid from the IAC column was omitted (see the Results and Discussion Section 3.1). Afterwards the fractions were cleaned-up and concentrated using 10 kDa cut-off centrifugal filter devices as described below (Section 2.2.4). This methodology was used to purify the serum PSA in a similar way. The serum sample treated with ethanolamine was injected into the anti-PSA column in a larger volume than the PSA standard. Thus, 10 g of PSA standard were diluted to 200 L with PBS and injected in the IAC system to test the influence of PSA concentration and of the injected sample volume on the PSA recovery in comparison with the one obtained for 20 L of PSA standard at 0.5 mg mL−1 .
2.2.4. Centrifugal filtration clean-up and concentration of PSA Clean-up and concentration of samples treated with ethanolamine or with propionic acid as well as the ones collected after elution from the anti-PSA column was carried out by ultrafiltration at 4 ◦ C using centrifugal filter devices with a 10 kDa ® cut-off membrane (Microcon YM-10). Prior to any PSA clean-up steps, ultrafiltration devices were ® treated with a 5% Brij35 solution for at least 12 h, in order to prevent the adsorption of the glycoprotein to the device [21]. The fractions containing PSA were washed with 300 L water four times at 14 000 × g for 30–35 min. More washing steps were performed when needed (according to the CZE profile obtained). Afterwards, the reservoir was turned upside down in a new vial and spun at 1 000 × g for 3 min. In this way the concentrated sample was recovered in 4–10 L water. The present work studied the influence of pH on PSA recovery ® from Microcon YM-10 devices by carrying out two sets of assays (see Table 1) in which different amounts of Tris base (1 M or 4 M) or 8 M propionic acid were added to PSA. In the first set (experiments 1–7 referred to as “in vial” in Table 1), PSA was diluted in solvents similar to the eluent of the IAC column. In experiments 5–7 PSA was dissolved in “blank eluate”; this solvent was obtained as the fraction eluted with 1 M propionic acid from the IAC column when PBS instead of PSA had been injected. In the second set of experiments (experiments 8–10, referred to as IAC in Table 1), the pH was modified on PSA that had been eluted with 1 M propionic acid from the anti-PSA column. The alkaline or acidic agents tested (“reagent added” column in Table 1) were added to the “in vial” PSA solutions or to the PSA eluted from the IAC column. In both sets of experiments, the PSA aliquots at different pH values were cleaned-up and concentrated on the centrifugal filter devices and analyzed by CZE to study the pattern and to calculate the recovery. For recovery calculation the total corrected area of PSA peaks in the CZE analysis was compared to that of untreated PSA standard; the sample volume (4–10 L) in which the sample was recovered after ® clean-up and concentration in the Microcon YM-10 devices was taken into account.
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Table 1 ® Influence of the pH on the recovery of PSA cleaned-up and concentrated on Microcon 10 centrifugal filter devices. EXPERIMENT 1 2 3 4 5 6 7 8 9 10 a b c d
METHOD In-vial In-vial In-vial In-vial In-vial In-vial In-vial IAC IAC IAC
PSA AMOUNT (g)
PSA SOLVENT
REAGENT ADDEDa
4.8 4.8 4.8 4.8 5.0 5.0 5.0 9.0 10.0 10.0
400 L 1 M propionic acid 400 L 1 M propionic acid 150 L PBS + 250 L 1 M propionic acid 150 L PBS + 250 L 1 M propionic acid 500 L Blank eluate (propionic acid + PBS) 500 L Blank eluate (propionic acid + PBS) 500 L Blank eluate (propionic acid + PBS) 450 L column eluate (propionic acid + PBS) 500 L column eluate (propionic acid + PBS) 500 L column eluate (propionic acid + PBS)
110 L 4 M Tris base 50 L 1 M Tris base 50 L 1 M Tris base – – 50 L 8 M propionic acid 120 L 8 M propionic acid 50 L 1 M Tris base – 120 L 8 M propionic acid
RECOVERY%b
pH c
7–8 5–6c 5c 3c 2.8 2.4 2.2 N.md 2.8 2.2
36.9 60.2 42.4 74.5 71.4 72.1 71.4 35.0 55.3 57.5
Reagent was added for pH adjustment prior to filtration. Recovery is expressed as percentage of total area of PSA standard measured by CZE. Approximated pH value measured by pH indicator test paper. Not measured.
2.2.5. Circular dichroism CD analysis of PSA standard was carried out at room temperature in the far UV region (260–190 nm) using a J-815 spectropolarimeter from JASCO (Tokyo, Japan). A quartz cell with 1 mm pathlength was used. CD spectra were recorded with a scan rate of 50 nm min−1 and a bandwidth of 1 nm. Measurements were carried out in triplicate and each of them was presented as average of four individual scans. CD analysis of PSA standard dissolved in water was performed as reference in each step of the method; that is, before and after ethanolamine treatment and after IAC. The CDPro software CONTINLL was used to analyze the protein CD spectra and to calculate the percentage of secondary structures. The “goodness of fit” parameter NRMSD (normalized root mean square deviation) is the most useful single measure of how well the theoretical CD spectrum calculated from the derived secondary structure composition matches the experimental data over the entire wavelength range of interest. As is common, the present work considered a value of NRMSD above 0.25 as constituting an error in the analysis procedure [22]. 2.2.6. Capillary zone electrophoresis CZE was carried out using a 7100 CE instrument from Agilent Technologies (Waghäusel, Germany), equipped with a UV–vis diode array detector. Capillaries employed were of 70 cm effective length and 78.5 cm total length, with 50 m I.D. A method recently optimized in our lab was employed [16]. Briefly, the capillary was conditioned initially and between-injections with HCl. The separation buffer consisted of sodium tetraborate (5 mM), sodium dihydrogen phosphate (10 mM), urea (3 M), and decamethonium bromide (2 mM), (pH 8.0). Separation voltage and temperature were 25 kV and 35 ◦ C, respectively. Sample injection was carried out at 35 mbar during 30 s, and UV detection was performed at 214 nm. Migration was expressed as effective electrophoretical mobility (eff ) because this parameter provides higher precision than migration time [16] 3. Results and discussion 3.1. Effect of acidity on PSA recovery and on CZE pattern after immunopurification, clean-up, and concentration Antigen elution from antibody IAC columns is usually performed by disrupting the immunorecognition forces using harsh conditions. When acidic or basic solutions are used as eluting agents, it is usually recommended to neutralize the eluted fraction as soon as possible to minimize protein denaturation. In our previous work we achieved this by eluting the PSA with propionic acid from the
affinity column and collecting it in 1 M Tris [17]. In the present work, the effect of modifying the pH of the collected PSA on recovery was determined by CZE. The design of the experiments took into account the fact that PSA is cleaned-up and concentrated using centrifugal filter devices between elution from the IAC column and injection on the CZE capillary. Therefore, the influence of the PSA ® solution pH on its recovery from the Microcon YM-10 devices was studied as described in the Experimental section. The first two experiments carried out “in-vial” by diluting PSA in 1 M propionic acid and adding different amounts of Tris base, showed that as the proportion of Tris base added increased (making the PSA solution more basic), the recovery of PSA from the filter device (Table 1) decreased. In the two following experiments (numbers 3 and 4 in Table 1), PSA was diluted in a PBS + propionic acid mixture in similar proportion and pH than a real fraction eluted from the IAC column. Adding Tris base until pH 5 (experiment 3) reduced the PSA recovery in comparison with the non-neutralized PSA solution filtered and concentrated at pH 3 (experiment 4). In order to determine whether decreasing the pH of the PSA solution prior ® to treatment in the Microcon YM-10 devices could improve the recovery, protein was solved in blank eluate and pH was modified by adding 8 M propionic acid (experiments 5–7). The acidification of the PSA solution to pH 2.4 or lower did not modify the amount of PSA recovered in comparison to pH 2.8, with the highest recoveries obtained under these acidic conditions (pHs 2.2-2.8). The “in-vial” experiments were corroborated with PSA injected into the anti-PSA column (experiments 8–10). For PSA submitted to immunorecognition and elution from the anti-PSA column, the recovery was 35% when 50 L of 1 M Tris were added to the eluted fraction prior to clean-up and concentration; and it increased by about 55% when the sample was treated in the centrifugal device without adding Tris (pH 2.8). Addition of 8 M propionic acid to the eluted PSA to ® decrease pH up to 2.2 prior to treatment in the Microcon device, did not markedly increase the PSA recovery. Furthermore, it was observed that clean-up and concentration in centrifugal devices led to CZE patterns of PSA that were not modified by prior addition of base or acid. Considering all these results, PSA eluted from the immunocolumn was not neutralized before being handled in ® Microcon devices for the rest of the study. Under these experimental conditions, comparison of the CE electropherograms of PSA that had been subjected to IAC at 0.5 mg mL−1 and at 0.05 mg mL−1 , indicated that recovery was not markedly affected by diluting 10 g PSA to 200 L with PBS prior to its injection in the anti-PSA column, and more importantly, similar CE profiles were obtained for both PSA standards (Supplementary information, Fig. S1).
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Fig. 1. Circular dichroism of PSA standard in each step of the sample preparation method including a final solvent exchange to water for the CD measurement. Far UV CD spectra for the steps of one sample preparation (left) and average and error bars (for 3 sample preparations) of secondary structure percentage (right) of (A) untreated PSA, (B) PSA treated with ethanolamine and (C) PSA treated with ethanolamine and subsequently IAC.
Fig. 2. CZE of PSA standard (A) without treatment, (B) treated with ethanolamine (ET) and cleaned in a centrifugal device (4 × 300 L water), (C) treated with ET, submitted to IAC, and insufficiently cleaned in a centrifugal device (4 × 300 L water), and (D) treated with ET, submitted to IAC, and extensively cleaned in a centrifugal device (4 × 300 L + 2 × 200 L water). Uncoated capillary (L = 78.5 cm, l = 70 cm, I.D. = 50 m). BGE: sodium tetraborate (5 mM), sodium dihydrogen phosphate (10 mM), decamethonium bromide (2 mM), urea (3 M), (pH 8.0). Separation at 25 kV and 35 ◦ C. Injection of PSA at 35 mbar for 30 s. Migration plotted as effective electrophoretic migration × 104 .
3.2. Effect of ethanolamine and its combination with immunopurification on fPSA: CD and CZE analyses Ethanolamine at alkaline pH has been employed to dissociate the PSA-ACT complex. After treatment with this nucleophilic agent the released PSA has been analyzed by 2-DE under denaturing conditions [18]. However, the CZE method used in the present study is performed under native conditions. To our knowledge, the effect of these dissociating conditions on the CZE profile of fPSA has not yet been studied. In order to do it, fPSA standard was treated (n = 3) with ethanolamine under the same conditions used to dissociate the complexed PSA in serum samples. The influence of the ethanolamine treatment and basic pH on fPSA was studied by CD and by CZE; each analysis was performed in triplicate. Prior to these analyses the dissociating agent was removed using 10 kDa cut-off centrifugal filter devices after the treatment. Comparison of the CD spectra and percentages of ␣ helix,  sheets, turns and unordered structures (Fig. 1) and of the CZE patterns (Fig. 2A and B) before and
after treatment with ethanolamine did not show marked changes in the protein configuration or the CZE pattern. In serum, fPSA has to be isolated from the matrix before performing CZE. The effect of the immunopurification step on ethanolamine-treated PSA is unknown. To test it, the fPSA standard sample that was treated with ethanolamine and further centrifugally filtered to remove the dissociating agent, was injected into the anti-PSA column and the retained fraction was eluted with 1 M propionic acid and subsequently cleaned-up and concentrated. Performing this filtration step as usual (4 × 300 L water) led to a CZE pattern that did not show any PSA peak (Fig. 2C). Our previous experience showed that the presence of some low molecular compounds in the PSA samples markedly altered the CE pattern (Supplementary information, Fig. S2). A more intense clean-up consisting in additional washing steps with water (extra 2 × 200 L water) allowed us to obtain a CZE pattern (Fig. 2D) that is highly repeatable (n = 3) in terms of peak size and migration (Supplementary information, Table S1), and in which the proportion of peaks was not markedly changed compared to the non-treated fPSA standard (Supplementary information, Fig. S3).The CD spectrum of this sample did not show displacement of the absorbance band in comparison with the untreated sample, indicating that the fPSA structure determined under the working conditions was not notably altered by being treated with ethanolamine and submitted to immunopurification under the same conditions that will be used for serum samples (Fig. 1). 3.3. CZE and 2DE analysis of PSA isoforms purified from a prostate cancer patient serum After proving that the above methodology did not drastically alter the CZE pattern for PSA standard, an experiment to study if the method was effective for isolating PSA from serum was performed. To differentiate the effect of the sample preparation from any other treatment (for example fluorescent labeling that would have been required to detect low PSA concentration) on the CZE pattern, the serum had to contain PSA at a concentration high enough to be detected by UV. Although very unusual, sera with PSA concentrations higher than 10 000 ng mL−1 have been found in some instances [23]. A serum sample with extremely high PSA concentration generously donated by a patient with advanced prostate cancer made it possible to perform the present study. After treatment with ethanolamine and removal of this reagent ® using Microcon YM-10 devices, the remaining 85 L of the treated clinical sample were diluted with PBS and injected into the antiPSA column. As expected, a large non-retained peak corresponding
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Fig. 3. CZE of serum PSA treated with ethanolamine and subsequently submitted to IAC. Comparison with PSA standard submitted to the same methodology and effect of repeated cleaning steps using Microcon devices. The rest of the conditions as in Fig. 2. (A) Serum PSA first cleaning, (B) serum PSA second cleaning, (C) serum PSA third cleaning and (D) PSA standard. CZE conditions as in Fig. 2. Peaks with the same number in figures (C) and (D) correspond to peaks with the same electrophoretic mobility. Peaks 6 and 7 in (C) have different mobility than any of the peaks in (D).
to the serum matrix was observed in the immunochromatogram. To wash out weakly retained potential impurities from the matrix, 1 mL of 1 M NaCl was injected at 0.5 mL min−1 into the column. This step had been shown not to elute PSA and to be effective in reducing interfering compounds from seminal plasma [17]. Accordingly, the non-retained fraction of this washing step did not show any peak in the zone of the electropherogram corresponding to PSA and showed a wide band at migration time about 38 min (data not shown) that could correspond to impurities removed from the sample. Next, a 1 M propionic acid elution step was performed and the eluted fraction from the IAC column, after being ® cleaned-up by centrifugation in a Microcon YM-10 device, was analyzed by CE (Fig. 3A). A disturbed pattern that did not resemble a PSA pattern was obtained. The migration times of the EOF marker (tEOF = 14.73 min) and of PSA peaks were too long and the resolution was too low in comparison with the CE behavior of PSA standard that had been submitted to the same treatment (Fig. 3D). An additional clean-up step with water in another centrifugal filter markedly improved the pattern of serum fPSA (Fig. 3B) and increased migration velocity (tEOF = 14.38 min). A third clean® up treatment in a third Microcon YM-10 device led to a clean pattern of PSA isoforms from serum (Fig. 3C) with migration (tEOF = 13.90 min) more similar to that of PSA standard analyzed on the same day (tEOF = 13.35 min). As seen in Fig. 3, the use of effective electrophoretic migration (eff ) instead of migration time (tm ) as the migration parameter made it possible to compare the electropherograms directly. In addition, as expected, better repeatability was achieved by expressing migration as eff (RSD < 1.4% even for the smallest peaks) rather than as tm (Supplementary information, Table S2). The SDS-PAGE and subsequent WB analysis of this purified PSA from the serum sample showed that treatment with ethanolamine effectively dissociated most of the complexed-PSA (Fig. 4, lane 3). According to Peter et al. [15], the low percentage of non-dissociated PSA could be due to incomplete cleavage of a low amount of PSAACT complex or to other PSA-serpin complexes present in serum at low concentration. The molecular size of the non-cleaved PSA observed by Western blot (Fig. 4, lane 3, band 2) could correspond to PSA-ACT as described recently [24] and not to the complex of PSA with alpha 2-macroglobulin, which would be larger. The major
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Fig. 4. PSA detection of serum and immunopurified PSA from the same serum in a 12% SDS-PAGE under reducing conditions, followed by Western blot with anti-PSA antibodies. Lane 1: molecular weight markers. Lane 2: 0.5 L serum, which contains 10 ng of total PSA. Lane 3: 25 ng of immunopurified PSA from serum. Lane 4: 100 ng of PSA standard. Band 1 corresponds to free PSA and band 2 to the ACT-PSA complex; nicked forms are also indicated.
band of immunopurified PSA corresponding to fPSA was observed at about 35 kDa, and bands of lower molecular weight corresponding to nicked forms were not found in the serum (Fig. 4, lane 2) or in PSA purified from it (Fig. 4, lane 3). However, some nicked forms were detected in PSA standard in a relatively lower proportion than the mature PSA (Fig. 4, lane 4). 2-DE followed by WB analysis showed that the sample treatment methodology including dissociation of complexed PSA and immunopurification did not modify the pattern of fPSA obtained from serum (Fig. 5A and B). The 2-DE pattern of the serum PSA before and after immunopurification showed the same proportion of the main F1-F4 subforms at around 33 kDa (Fig. 5C). Nicked forms were very minor and hardly detected, in agreement with the results of the SDS-PAGE analysis of serum PSA mentioned above (Fig. 4). The conditions of 2-DE used for PSA analysis do not show the PSA complexed to ACT from the serum sample that focuses at higher molecular weight and at more acid pH as described previously [25]. fPSA 2-DE pattern is sensitive to PSA glycosylation changes [12] and to other post-translational modifications that could lead to variations in PSA pI and/or molecular weight. Therefore, the dissociation and immunopurification methodology described here allowed purifying the same PSA 2-DE subforms found in the original serum PSA, without selecting or selectively enriching any of them. The different CZE patterns of serum and seminal plasma PSA, both submitted to the sample treatment methodology developed, could be due to several reasons. These differences could reflect changes in the peptidic chain linkages (nicked forms), changes in glycosylation or in other types of modifications (phosphorylation, sulfation, etc) as a result of cancer. It has to be taken into account that the serum PSA was obtained from a patient with very advanced prostate cancer, while the commercial PSA standard was prepared from the seminal plasma of healthy donors. Regarding glycosylation, differences between the CZE profiles of serum PSA (Fig. 3C) and the PSA standard from seminal plasma (Fig. 2A) could be related to the differences in fucosylation and N-acetyl galactosamine content as well as in the linkage of sialic acid between PSA from a
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Fig. 5. 2-DE and Western blot analysis of PSA. (A) 2-DE pattern of 1 uL of serum of a prostate cancer patient, containing 20 ng of PSA, (B) 2-DE pattern of 15 ng of PSA purified from the same serum of a prostate cancer patient, (C) densitometry profile of the F1-F4 PSA spots.
cancer patient’s serum and from a control seminal plasma previously found using different analytical techniques [11,23,26]. Also it is possible that these differences in CZE patterns between samples arise from the fact that seminal plasma PSA is present only as fPSA, while the PSA purified from serum contains the original fPSA plus that released from the ACT-PSA complex. 4. Concluding remarks A method that makes it possible to purify PSA from serum in a way that is compatible with analysis of isoforms of the glycoprotein by CZE was developed. The sample preparation methodology necessary to analyze fPSA in serum samples consisting of treatment with ethanolamine and immunochromatographic purification does not notably alter the CD spectrum or the CE-UV profile of PSA. In addition, the 2-DE pattern of serum PSA is not modified by the sample treatment. Thorough removal of the reagents used in the sample preparation process is mandatory prior to the CE analysis for avoiding disturbances. Under the optimized sample preparation conditions, highly repetitive CD and CZE results are obtained. The CZE pattern of PSA from a serum sample of a prostate cancer patient show differences in comparison to the pattern of PSA standard from seminal plasma of healthy donors. These differences, that may reflect changes in glycosylation or in other features of the molecule, could arise either from the clinical stage of the donors or from the nature of the fluid. This analysis methodology, which includes sample preparation and capillary electrophoresis separation of PSA isoforms, makes it feasible to analyze the isoform pattern of this glycoprotein in rich PSA serum samples. In the future, CZE patterns of serum PSA from individuals with different clinical conditions (healthy, prostate cancer patients with different Gleason scores, and patients with non-malignant prostate diseases) could be compared to evaluate the usefulness of the CZE pattern as a prostate cancer marker.
For samples with lower PSA concentration, CE detection methods with enhanced sensitivity that maintain the separation of isoforms will be required. Work in this direction is currently being carried out in our laboratory. Acknowledgements The generous gift of the serum sample by the cancer patient donor is highly appreciated. We would also like to acknowledge the financial support from the Spanish Ministry of Economy and Competitiveness (MINECO, projects CTQ2013-43236-R, BIO201016922, and BIO2015-66356-R) and from the Generalitat de Catalunya, Spain (grant 2014 SGR 229). N. F.-G. and M. G. acknowledge the Ph.D. JAE-pre grant and the JAE-doc grant, respectively, from CSIC co-financed by the European Social Fund. D.N-C. acknowledges the contract in the frame of the Youth Guarantee Implementation Plans financed by the European Social Fund (ESF) and the Youth Employment Initiative (YEI). J.E. G.-L. acknowledges support by Award Number R01GM089759 from the National Institute of General Medical Sciences (NIH, U.S.A.). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jpba.2016.11.045. References [1] L.A. Torre, F. Bray, R.L. Siegel, J. Ferlay, J. Lortet-Tieulent, A. Jemal, Global cancer statistics, 2012, CA—Cancer J. Clin. 65 (2) (2015) 87–108. [2] R.L. Siegel, K.D. Miller, A. Jemal, Cancer statistics, 2015, CA—Cancer J. Clin. 65 (1) (2015) 5–29. [3] E.H. Kim, G.L. Andriole, Prostate-specific antigen-based screening: controversy and guidelines, BMC Med. 13 (2015). [4] J. Cuzick, M.A. Thorat, G. Andriole, O.W. Brawley, P.H. Brown, Z. Culig, R.A. Eeles, L.G. Ford, F.C. Hamdy, L. Holmberg, D. Ilic, T.J. Key, C. La Vecchia, H. Lilja, M. Marberger, F.L. Meyskens, L.M. Minasian, C. Parker, H.L. Parnes, S. Perner, H. Rittenhouse, J. Schalken, H.-P. Schmid, B.J. Schmitz-Draeger, F.H. Schroder,
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Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Sample preparation of serum to allow capillary electrophoresis analysis of prostate specific antigen isoforms Noemi Farina-Gomez a , Silvia Barrabes b , Jorge E. Gomez-Lopez a,c,1 , Monica Gonzalez a , Angel Puerta a , Diana Navarro-Calderon a , Eduardo Albers-Acosta d , Carlos Olivier d , Jose C. Diez-Masa a , Rosa Peracaula b,∗ , Mercedes de Frutos a,∗∗ a
Institute of Organic Chemistry (IQOG-CSIC), Madrid, Spain Unit of Biochemistry, Faculty of Sciences, University of Girona, Girona, Spain c Department of Chemistry, Universidad del Valle, Cali, Colombia d Department of Urology, Hospital La Princesa, Madrid, Spain b
a r t i c l e
i n f o
Article history: Received 28 July 2016 Received in revised form 21 November 2016 Accepted 23 November 2016 Available online 24 November 2016 Keywords: Sample treatment Serum Prostate specific antigen Capillary electrophoresis Two-dimensional electrophoresis Prostate cancer biomarker
a b s t r a c t Prostate cancer is the second most frequently diagnosed cancer in men worldwide. Currently prostate specific antigen (PSA) serum concentration is the most used prostate cancer marker, but it only shows limited specificity. Because PSA glycosylation is altered by prostate cancer, detecting glycosylation changes could increase PSA specificity as a prostate cancer marker. Changes in PSA glycosylation can modify its electrophoretic- behavior and techniques such as capillary zone electrophoresis (CZE) and two-dimensional electrophoresis (2-DE) could be applied to detect changes in PSA glycosylation. Most serum PSA is complexed with alpha-1 antichymotrypsin (ACT). To have access to most of the PSA, the complexed PSA has to be released as free PSA (fPSA); in addition, this total fPSA must be purified from the serum matrix so that it can be analyzed using CZE. In this work a methodology for isolating PSA from serum for its CZE analysis was established. By using PSA standard, the effect of this methodology, which combines conditions for dissociating complexed PSA and immunoaffinity chromatographic purification, was studied. It was seen that this highly repeatable sample treatment did not noticeably alter the circular dichroism (CD) spectrum or the CZE pattern of PSA standard. Therefore, as a proof-of-concept, the developed sample treatment was applied to serum from a cancer patient with a high PSA content. The following observations can be made from these experiments: first of all, the 2-DE pattern of serum PSA remained unchanged after sample treatment; second, as hypothesized, the established sample preparation methodology made it possible to obtain the CZE pattern of PSA from serum; and third, the CZE pattern of serum PSA and of PSA standard from seminal plasma of healthy individuals, both submitted to the sample treatment method, showed some differences regarding the proportion of CZE peaks of the glycoprotein. These differences could be related to possible changes in the linkages of peptide backbone, in glycosylation or in other post-translational modifications between samples from both origins. © 2016 Elsevier B.V. All rights reserved.
1. Introduction
Abbreviations: ACT, alpha-1 antichymotrypsin; 2-DE, two-dimensional electrophoresis; fPSA, free PSA; HRP, horseradish peroxidase; IAC, immunoaffinity chromatography; PSA, prostate specific antigen; tPSA, total PSA; Tris, Tris (hydroxymethyl) aminomethane; WB, Western blot. ∗ Corresponding author at: Biology Department, Faculty of Sciences, University of Girona, 17071, Girona, Spain. ∗∗ Corresponding author at: Department of Instrumental Analysis and Environmental Chemistry, Institute of Organic Chemistry, Spanish Research Council (IQOG- CSIC), Juan de la Cierva 3, 28006, Madrid, Spain. E-mail addresses: [email protected] (R. Peracaula), [email protected] (M. de Frutos). 1 Present address: Department of Chemistry, Universidad Nacional de Colombia, Bogotá, Colombia. http://dx.doi.org/10.1016/j.jpba.2016.11.045 0731-7085/© 2016 Elsevier B.V. All rights reserved.
In 2012, over 14 million new cancer cases and more than 8 million cancer deaths were reported worldwide. Out of them, 1.1 million corresponded to new cases of prostate cancer, which is the second most frequently diagnosed cancer in men worldwide [1]. While five-year survival for prostate cancer is estimated to be >99% for localized cancer, survival markedly decreases to less than 30% for distant cancer [2]. Controversial results on how cancer mortality is affected by screening based on serum prostate specific antigen (PSA) concentration are being published [3]. Its main drawback is its limited specificity, since non-malignant prostate diseases can also increase
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the level of serum PSA. Thus, the need for improved prostate cancer diagnostic methods has been emphasized [4]. In addition to genomic, proteomic, and metabolomic advances in the search for new biomarkers, approaches based on glycomics and glycoproteomics are also being applied because changes in glycosylation have been associated with cancer development and progression [5,6]. Specifically, modifications in the glycosylation of PSA and other proteins have been described in prostate cancer [7–10] and some of them, in particular core fucosylation levels and percentage of ␣2,3 sialic acid, have recently been described to identify aggressive prostate cancer and could, therefore, be useful to improve prostate cancer diagnosis [11]. Changes in glycosylation can alter the size and/or charge of the different molecular forms of a given glycoprotein and, therefore, their migration when analyzed by electrophoretic techniques such as two-dimensional electrophoresis (2-DE) or capillary electrophoresis (CE) can also be altered. 2-DE analyses of PSA have shown that the obtained PSA spot pattern can be partially explained by the different sialic acid content of PSA N-glycan chains [12]. However, techniques that are faster, more quantitative and easier to automate than 2-DE are desirable to reveal changes in the electrophoretic pattern of PSA. CE has these qualities and using this technique the patterns of some non-hydrolyzed glycoproteins have been shown to be potential disease biomarkers [13,14]. PSA is present in serum as a complex mixture of several species. The most abundant form is a covalent complex of PSA with alpha-1 antichymotrypsin (ACT) through an ester linkage. Free PSA (fPSA), including nicked forms corresponding to internally cleaved PSA polypeptide chains, accounts for only 5–30% of the total PSA (tPSA) [15]. In order to investigate if the CE pattern of serum PSA could be useful as a prostate cancer biomarker it is critical to use appropriate CE and sample preparation methods. The CE method should be able to separate PSA isoforms (peaks containing one or more glycoforms of fPSA). Such a CE method has been recently optimized by our group [16]. The sample preparation method should provide a high purity of the glycoprotein from the biological fluid without altering its CE profile. However, a serum treatment that makes it possible to purify PSA from it in a way that is compatible with CE analysis is not available. An immunoaffinity chromatography (IAC) method has been developed for isolating PSA from seminal plasma, where it exists as fPSA at high concentration [17]. However, most of PSA in serum exists as complexed PSA and its concentration is much lower than in seminal plasma. To have access to most of the tPSA, which can be more representative of modifications that take place due to prostate cancer [18], and to increase the available concentration of fPSA to be analyzed by CE, the ACT-PSA complex has to be dissociated. A method that employs ethanolamine to release PSA from this complex has already been described [15]. This method has been applied with some modifications to release PSA for 2-DE analysis under reducing conditions [18]. The CE methods developed to analyze fPSA isoforms work under non-reducing conditions [16,19,20]. Therefore, the five disulphide bridges of PSA held the protein structure together, including the nicked forms. This means that PSA isoform profile will be different if the analysis methods are performed under reducing conditions or under non-reducing ones. PSA released from serum by ethanolamine treatment was found to be very similar to PSA standard when using immunoassays, HPLC and MS [15]. However, the authors of this treatment indicated that it would be interesting to perform some experiments, including immunorecognition, regarding the structural integrity of PSA dissociated from the complex. The feasibility of purifying PSA from serum using an antiPSA affinity column, previously described for seminal plasma PSA
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purification, has not been addressed. In this regard, the effect of ethanolamine treatment on PSA recognition by the anti-PSA antibodies of the immunocolumn needs to be tested. In addition, the influence of a different matrix composition and of a much larger sample volume to compensate for the low PSA concentration in serum has not been studied. Furthermore, the effect of ethanolamine in combination with immunopurification on PSA structure and on its capillary electrophoresis pattern under nonreducing conditions has not been addressed. These questions, which must be resolved in order to validate the usefulness of the CE pattern of PSA as prostate cancer marker in the future, are studied in the present work. Specifically, a methodology to treat PSA with the dissociating agent and to purify the free glycoprotein using a house-made anti-PSA column that preserves separation of PSA isoforms by CE was developed using fPSA standard. SDS-PAGE followed by Western blot (WB), CD and CZE were used to control possible protein alterations during the different steps of the method. Finally, the usefulness of the sample preparation methodology for a clinical sample was tested by applying it to a prostate cancer patient’s serum and analyzing PSA from this fluid by CE and 2-DE.
2. Experimental 2.1. Samples, reagents, and devices 2.1.1. Materials ® The European standard (Certified Reference Material BCR 613) of fPSA purified from human seminal plasma used to develop the methodology was acquired from Isostandards Material (Madrid, Spain). The monoclonal (clone 5G6) anti-tPSA antibody from Antibodies on-line (Aachen, Germany) as well as the rest of materials used to fabricate the 3 cm x 2.1 mm I.D. anti-PSA column were as previously described [17]. Tris (hydroxymethyl) ® aminomethane (Tris), urea, Brij35 , propionic acid, sodium tetraborate, and decamethonium bromide were from Sigma-Aldrich (Steinheim, Germany). Sodium azide was from J.T. Baker (London, UK). Hydrochloric acid was from Panreac (Barcelona, Spain). Ethanolamine was from Fluka (Steinheim, Germany). Disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, and potassium chloride were from Merck (Darmstadt, Germany). The phosphate buffered saline (PBS) consisted of disodium hydrogen phosphate (0.1 M)/sodium dihydrogen phosphate (0.1 M) buffer with NaCl (1.38 M) and KCl (27 mM) (pH 7.4). ® Microcon YM-10 devices were from Millipore (Billerica, MA, USA). Fused silica capillaries were provided by CM Scientific Limited (Silsden, UK). The following reagents were used to perform SDS-PAGE, 2DE and Western blot (WB): 40% polyacrylamide solution and horseradish peroxidase (HRP) conjugated goat anti-rabbit IgG (H + L) were from Thermo Fisher Scientific (Waltham, MA, USA). Tris, Tris-HCl, glycine, SDS, ß-mercaptoethanol, bromophenol blue, urea and Triton X-100 were from Serva (Heidelberg, Germany). Glycerol and bovine serum albumin (BSA) were from Roche Diagnostics (Mannheim, Germany). PVDF membranes and Immobilon Western chemiluminescent HRP substrate were from Millipore. Methanol was from Panreac. Tween-20 was from Sigma-Aldrich. Dithiothreitol (DTT), iodoacetamide, pharmalyte 3–10 and Immobiline DryStrip gels (IPG strips) 18 cm pH 3–10 linear were from GE Healthcare (Buckinghamshire, UK). Agarose was from Ecogen (Barcelona, Spain) and polyclonal rabbit anti-human PSA was from Dako (Glostrup, Denmark). In all the experiments water was of milli-Q quality and it was obtained from a Millipore purification system (Bedford, MA).
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A serum sample from a prostate cancer patient with very high PSA concentration (>20 000 ng mL−1 ) was obtained at the Hospital de la Princesa (Madrid, Spain) following the guidelines of the hospital’s Ethical Committee. 2.1.2. Sample handling Lyophilized European PSA standard was dissolved in water (1 mg mL−1 ), aliquoted, and stored at −20 ◦ C until use. For SDSPAGE, it was diluted 1:9 (v/v) in water and 100 ng were loaded in Laemmli buffer (Tris/HCl (30 mM), SDS (2%, w/v), glycerol (10%, v/v), bromophenol blue (0.0125%, w/v), (pH 6.8)) with mercaptoethanol (1.25%, v/v); for IAC, it was diluted 1:1 (v/v) with 2× concentrated PBS. The serum sample was aliquoted and kept at −20 ◦ C until use. 2.2. Methods and equipment 2.2.1. Dissociation of PSA-ACT complex Treatment with ethanolamine was performed according to Peter et al. [15]. In short, 200 L PSA standard (30 g) or 175 L serum sample were treated with 2 M ethanolamine in PBS to get a 0.1 M final concentration of ethanolamine. The mixture was adjusted to pH 10.3 with HCl and incubated at 25 ◦ C for 24 h. Before PSA isolation, the reaction was stopped by removing ethanolamine using centrifugal filter devices with a 10 kDa cut-off membrane (Micro® con YM-10). 2.2.2. Polyacrylamide gel electrophoresis (1-DE and 2-DE) and western blot to immunodetect PSA SDS-PAGE was performed using a 12% polyacrilamide gel in resolving buffer (Tris-HCl (pH 8.8, 1.5 M), SDS (0.4%)) and a 5% polyacrilamide gel in stacking buffer (Tris-HCl (pH 6.8, 0.5 M), SDS (0.4%)). Samples were prepared with Laemmli buffer. Gel was run for 2 h at 100 V in running buffer (glycine (192 mM), Tris-HCl (25 mM), (pH 8.4), SDS (0.1%)), and transferred to a PVDF membrane for 3 h at 100 V in Towbin buffer (Tris (25 mM), glycine (192 mM), (pH 8.3), methanol (20%)). The membrane was blocked with a 3% BSA solution in Tris buffered saline-Tween (TBST) (Tris (10 mM), NaCl (100 mM), Tween-20 (0.1%)). PSA was detected using a polyclonal rabbit anti-PSA antibody diluted 1/4000 in 0.5% BSA in TBST, followed by a secondary goat anti-rabbit horseradish peroxidaseconjugated antibody diluted 1/80000 in 0.5% BSA in TBST. Several washing steps with TBST were performed between each step. Detection was performed using the Immobilon Western Chemiluminescent HRP Substrate and chemiluminescence was visualized using the Fluorochem SP imaging system (Alpha Innotech, San Leandro, CA, USA) under non-saturating conditions. Densitometry analysis was performed with AlphaEase FC Imaging system software (Alpha Innotech). 2-DE was performed as described previously [18] with minor modifications: In brief, samples (serum and PSA purified from the same serum) were diluted in 360 L of rehydration buffer (urea (8 M), Triton X-100 (0.5%), DTT (13 mM), Pharmalyte 3–10 (1%)) and were loaded in IPG strips pH 3–10, 18 cm by active in-gel rehydration. Isoelectric focusing was performed for a total run of 47 kV h. After the isoelectric focusing, the strips were cut to 7 cm keeping just the 5.5–7.5 pH range. For the second dimension the strips were first equilibrated for 15 min in equilibration buffer (urea (6 M), glycerol (30%, v/v), SDS (2%), Tris (50 mM), (pH 8.8)) containing 65 mM DTT and afterwards for 15 min in equilibration buffer containing 135 mM iodoacetamide. The strips were then placed on top of 10% polyacrylamide gels and sealed with a 0.5% agarose solution in running buffer (glycine (192 mM), Tris-HCl (25 mM), (pH 8.3–8.5), SDS (0.1%)) and traces of bromophenol blue. The second dimension was performed in a Miniprotean III unit (Bio-Rad, Hercules, CA, USA) with the running buffer described above and run at 1 W per gel.
Proteins were then transferred on a PVDF membrane and Western blot was carried out as described above.
2.2.3. Immunoaffinity isolation The method used was slightly modified from the one previously developed [17]. Briefly, PBS was pumped through the IAC column at 0.5 mL min−1 for 20 min before sample injection. Then, PSA standard was injected into the IAC column. PBS (0.5 mL min−1 ) was used as mobile phase. When baseline was recovered, 1 mL of 1 M propionic acid was injected into the column in order to elute the retained PSA and to collect it. Elution with propionic acid was carried out in triplicate, and the eluted fractions were collected on 1 M Tris. This method was modified throughout the present work, and the addition of 1 M Tris to the fraction eluted with propionic acid from the IAC column was omitted (see the Results and Discussion Section 3.1). Afterwards the fractions were cleaned-up and concentrated using 10 kDa cut-off centrifugal filter devices as described below (Section 2.2.4). This methodology was used to purify the serum PSA in a similar way. The serum sample treated with ethanolamine was injected into the anti-PSA column in a larger volume than the PSA standard. Thus, 10 g of PSA standard were diluted to 200 L with PBS and injected in the IAC system to test the influence of PSA concentration and of the injected sample volume on the PSA recovery in comparison with the one obtained for 20 L of PSA standard at 0.5 mg mL−1 .
2.2.4. Centrifugal filtration clean-up and concentration of PSA Clean-up and concentration of samples treated with ethanolamine or with propionic acid as well as the ones collected after elution from the anti-PSA column was carried out by ultrafiltration at 4 ◦ C using centrifugal filter devices with a 10 kDa ® cut-off membrane (Microcon YM-10). Prior to any PSA clean-up steps, ultrafiltration devices were ® treated with a 5% Brij35 solution for at least 12 h, in order to prevent the adsorption of the glycoprotein to the device [21]. The fractions containing PSA were washed with 300 L water four times at 14 000 × g for 30–35 min. More washing steps were performed when needed (according to the CZE profile obtained). Afterwards, the reservoir was turned upside down in a new vial and spun at 1 000 × g for 3 min. In this way the concentrated sample was recovered in 4–10 L water. The present work studied the influence of pH on PSA recovery ® from Microcon YM-10 devices by carrying out two sets of assays (see Table 1) in which different amounts of Tris base (1 M or 4 M) or 8 M propionic acid were added to PSA. In the first set (experiments 1–7 referred to as “in vial” in Table 1), PSA was diluted in solvents similar to the eluent of the IAC column. In experiments 5–7 PSA was dissolved in “blank eluate”; this solvent was obtained as the fraction eluted with 1 M propionic acid from the IAC column when PBS instead of PSA had been injected. In the second set of experiments (experiments 8–10, referred to as IAC in Table 1), the pH was modified on PSA that had been eluted with 1 M propionic acid from the anti-PSA column. The alkaline or acidic agents tested (“reagent added” column in Table 1) were added to the “in vial” PSA solutions or to the PSA eluted from the IAC column. In both sets of experiments, the PSA aliquots at different pH values were cleaned-up and concentrated on the centrifugal filter devices and analyzed by CZE to study the pattern and to calculate the recovery. For recovery calculation the total corrected area of PSA peaks in the CZE analysis was compared to that of untreated PSA standard; the sample volume (4–10 L) in which the sample was recovered after ® clean-up and concentration in the Microcon YM-10 devices was taken into account.
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Table 1 ® Influence of the pH on the recovery of PSA cleaned-up and concentrated on Microcon 10 centrifugal filter devices. EXPERIMENT 1 2 3 4 5 6 7 8 9 10 a b c d
METHOD In-vial In-vial In-vial In-vial In-vial In-vial In-vial IAC IAC IAC
PSA AMOUNT (g)
PSA SOLVENT
REAGENT ADDEDa
4.8 4.8 4.8 4.8 5.0 5.0 5.0 9.0 10.0 10.0
400 L 1 M propionic acid 400 L 1 M propionic acid 150 L PBS + 250 L 1 M propionic acid 150 L PBS + 250 L 1 M propionic acid 500 L Blank eluate (propionic acid + PBS) 500 L Blank eluate (propionic acid + PBS) 500 L Blank eluate (propionic acid + PBS) 450 L column eluate (propionic acid + PBS) 500 L column eluate (propionic acid + PBS) 500 L column eluate (propionic acid + PBS)
110 L 4 M Tris base 50 L 1 M Tris base 50 L 1 M Tris base – – 50 L 8 M propionic acid 120 L 8 M propionic acid 50 L 1 M Tris base – 120 L 8 M propionic acid
RECOVERY%b
pH c
7–8 5–6c 5c 3c 2.8 2.4 2.2 N.md 2.8 2.2
36.9 60.2 42.4 74.5 71.4 72.1 71.4 35.0 55.3 57.5
Reagent was added for pH adjustment prior to filtration. Recovery is expressed as percentage of total area of PSA standard measured by CZE. Approximated pH value measured by pH indicator test paper. Not measured.
2.2.5. Circular dichroism CD analysis of PSA standard was carried out at room temperature in the far UV region (260–190 nm) using a J-815 spectropolarimeter from JASCO (Tokyo, Japan). A quartz cell with 1 mm pathlength was used. CD spectra were recorded with a scan rate of 50 nm min−1 and a bandwidth of 1 nm. Measurements were carried out in triplicate and each of them was presented as average of four individual scans. CD analysis of PSA standard dissolved in water was performed as reference in each step of the method; that is, before and after ethanolamine treatment and after IAC. The CDPro software CONTINLL was used to analyze the protein CD spectra and to calculate the percentage of secondary structures. The “goodness of fit” parameter NRMSD (normalized root mean square deviation) is the most useful single measure of how well the theoretical CD spectrum calculated from the derived secondary structure composition matches the experimental data over the entire wavelength range of interest. As is common, the present work considered a value of NRMSD above 0.25 as constituting an error in the analysis procedure [22]. 2.2.6. Capillary zone electrophoresis CZE was carried out using a 7100 CE instrument from Agilent Technologies (Waghäusel, Germany), equipped with a UV–vis diode array detector. Capillaries employed were of 70 cm effective length and 78.5 cm total length, with 50 m I.D. A method recently optimized in our lab was employed [16]. Briefly, the capillary was conditioned initially and between-injections with HCl. The separation buffer consisted of sodium tetraborate (5 mM), sodium dihydrogen phosphate (10 mM), urea (3 M), and decamethonium bromide (2 mM), (pH 8.0). Separation voltage and temperature were 25 kV and 35 ◦ C, respectively. Sample injection was carried out at 35 mbar during 30 s, and UV detection was performed at 214 nm. Migration was expressed as effective electrophoretical mobility (eff ) because this parameter provides higher precision than migration time [16] 3. Results and discussion 3.1. Effect of acidity on PSA recovery and on CZE pattern after immunopurification, clean-up, and concentration Antigen elution from antibody IAC columns is usually performed by disrupting the immunorecognition forces using harsh conditions. When acidic or basic solutions are used as eluting agents, it is usually recommended to neutralize the eluted fraction as soon as possible to minimize protein denaturation. In our previous work we achieved this by eluting the PSA with propionic acid from the
affinity column and collecting it in 1 M Tris [17]. In the present work, the effect of modifying the pH of the collected PSA on recovery was determined by CZE. The design of the experiments took into account the fact that PSA is cleaned-up and concentrated using centrifugal filter devices between elution from the IAC column and injection on the CZE capillary. Therefore, the influence of the PSA ® solution pH on its recovery from the Microcon YM-10 devices was studied as described in the Experimental section. The first two experiments carried out “in-vial” by diluting PSA in 1 M propionic acid and adding different amounts of Tris base, showed that as the proportion of Tris base added increased (making the PSA solution more basic), the recovery of PSA from the filter device (Table 1) decreased. In the two following experiments (numbers 3 and 4 in Table 1), PSA was diluted in a PBS + propionic acid mixture in similar proportion and pH than a real fraction eluted from the IAC column. Adding Tris base until pH 5 (experiment 3) reduced the PSA recovery in comparison with the non-neutralized PSA solution filtered and concentrated at pH 3 (experiment 4). In order to determine whether decreasing the pH of the PSA solution prior ® to treatment in the Microcon YM-10 devices could improve the recovery, protein was solved in blank eluate and pH was modified by adding 8 M propionic acid (experiments 5–7). The acidification of the PSA solution to pH 2.4 or lower did not modify the amount of PSA recovered in comparison to pH 2.8, with the highest recoveries obtained under these acidic conditions (pHs 2.2-2.8). The “in-vial” experiments were corroborated with PSA injected into the anti-PSA column (experiments 8–10). For PSA submitted to immunorecognition and elution from the anti-PSA column, the recovery was 35% when 50 L of 1 M Tris were added to the eluted fraction prior to clean-up and concentration; and it increased by about 55% when the sample was treated in the centrifugal device without adding Tris (pH 2.8). Addition of 8 M propionic acid to the eluted PSA to ® decrease pH up to 2.2 prior to treatment in the Microcon device, did not markedly increase the PSA recovery. Furthermore, it was observed that clean-up and concentration in centrifugal devices led to CZE patterns of PSA that were not modified by prior addition of base or acid. Considering all these results, PSA eluted from the immunocolumn was not neutralized before being handled in ® Microcon devices for the rest of the study. Under these experimental conditions, comparison of the CE electropherograms of PSA that had been subjected to IAC at 0.5 mg mL−1 and at 0.05 mg mL−1 , indicated that recovery was not markedly affected by diluting 10 g PSA to 200 L with PBS prior to its injection in the anti-PSA column, and more importantly, similar CE profiles were obtained for both PSA standards (Supplementary information, Fig. S1).
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Fig. 1. Circular dichroism of PSA standard in each step of the sample preparation method including a final solvent exchange to water for the CD measurement. Far UV CD spectra for the steps of one sample preparation (left) and average and error bars (for 3 sample preparations) of secondary structure percentage (right) of (A) untreated PSA, (B) PSA treated with ethanolamine and (C) PSA treated with ethanolamine and subsequently IAC.
Fig. 2. CZE of PSA standard (A) without treatment, (B) treated with ethanolamine (ET) and cleaned in a centrifugal device (4 × 300 L water), (C) treated with ET, submitted to IAC, and insufficiently cleaned in a centrifugal device (4 × 300 L water), and (D) treated with ET, submitted to IAC, and extensively cleaned in a centrifugal device (4 × 300 L + 2 × 200 L water). Uncoated capillary (L = 78.5 cm, l = 70 cm, I.D. = 50 m). BGE: sodium tetraborate (5 mM), sodium dihydrogen phosphate (10 mM), decamethonium bromide (2 mM), urea (3 M), (pH 8.0). Separation at 25 kV and 35 ◦ C. Injection of PSA at 35 mbar for 30 s. Migration plotted as effective electrophoretic migration × 104 .
3.2. Effect of ethanolamine and its combination with immunopurification on fPSA: CD and CZE analyses Ethanolamine at alkaline pH has been employed to dissociate the PSA-ACT complex. After treatment with this nucleophilic agent the released PSA has been analyzed by 2-DE under denaturing conditions [18]. However, the CZE method used in the present study is performed under native conditions. To our knowledge, the effect of these dissociating conditions on the CZE profile of fPSA has not yet been studied. In order to do it, fPSA standard was treated (n = 3) with ethanolamine under the same conditions used to dissociate the complexed PSA in serum samples. The influence of the ethanolamine treatment and basic pH on fPSA was studied by CD and by CZE; each analysis was performed in triplicate. Prior to these analyses the dissociating agent was removed using 10 kDa cut-off centrifugal filter devices after the treatment. Comparison of the CD spectra and percentages of ␣ helix,  sheets, turns and unordered structures (Fig. 1) and of the CZE patterns (Fig. 2A and B) before and
after treatment with ethanolamine did not show marked changes in the protein configuration or the CZE pattern. In serum, fPSA has to be isolated from the matrix before performing CZE. The effect of the immunopurification step on ethanolamine-treated PSA is unknown. To test it, the fPSA standard sample that was treated with ethanolamine and further centrifugally filtered to remove the dissociating agent, was injected into the anti-PSA column and the retained fraction was eluted with 1 M propionic acid and subsequently cleaned-up and concentrated. Performing this filtration step as usual (4 × 300 L water) led to a CZE pattern that did not show any PSA peak (Fig. 2C). Our previous experience showed that the presence of some low molecular compounds in the PSA samples markedly altered the CE pattern (Supplementary information, Fig. S2). A more intense clean-up consisting in additional washing steps with water (extra 2 × 200 L water) allowed us to obtain a CZE pattern (Fig. 2D) that is highly repeatable (n = 3) in terms of peak size and migration (Supplementary information, Table S1), and in which the proportion of peaks was not markedly changed compared to the non-treated fPSA standard (Supplementary information, Fig. S3).The CD spectrum of this sample did not show displacement of the absorbance band in comparison with the untreated sample, indicating that the fPSA structure determined under the working conditions was not notably altered by being treated with ethanolamine and submitted to immunopurification under the same conditions that will be used for serum samples (Fig. 1). 3.3. CZE and 2DE analysis of PSA isoforms purified from a prostate cancer patient serum After proving that the above methodology did not drastically alter the CZE pattern for PSA standard, an experiment to study if the method was effective for isolating PSA from serum was performed. To differentiate the effect of the sample preparation from any other treatment (for example fluorescent labeling that would have been required to detect low PSA concentration) on the CZE pattern, the serum had to contain PSA at a concentration high enough to be detected by UV. Although very unusual, sera with PSA concentrations higher than 10 000 ng mL−1 have been found in some instances [23]. A serum sample with extremely high PSA concentration generously donated by a patient with advanced prostate cancer made it possible to perform the present study. After treatment with ethanolamine and removal of this reagent ® using Microcon YM-10 devices, the remaining 85 L of the treated clinical sample were diluted with PBS and injected into the antiPSA column. As expected, a large non-retained peak corresponding
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Fig. 3. CZE of serum PSA treated with ethanolamine and subsequently submitted to IAC. Comparison with PSA standard submitted to the same methodology and effect of repeated cleaning steps using Microcon devices. The rest of the conditions as in Fig. 2. (A) Serum PSA first cleaning, (B) serum PSA second cleaning, (C) serum PSA third cleaning and (D) PSA standard. CZE conditions as in Fig. 2. Peaks with the same number in figures (C) and (D) correspond to peaks with the same electrophoretic mobility. Peaks 6 and 7 in (C) have different mobility than any of the peaks in (D).
to the serum matrix was observed in the immunochromatogram. To wash out weakly retained potential impurities from the matrix, 1 mL of 1 M NaCl was injected at 0.5 mL min−1 into the column. This step had been shown not to elute PSA and to be effective in reducing interfering compounds from seminal plasma [17]. Accordingly, the non-retained fraction of this washing step did not show any peak in the zone of the electropherogram corresponding to PSA and showed a wide band at migration time about 38 min (data not shown) that could correspond to impurities removed from the sample. Next, a 1 M propionic acid elution step was performed and the eluted fraction from the IAC column, after being ® cleaned-up by centrifugation in a Microcon YM-10 device, was analyzed by CE (Fig. 3A). A disturbed pattern that did not resemble a PSA pattern was obtained. The migration times of the EOF marker (tEOF = 14.73 min) and of PSA peaks were too long and the resolution was too low in comparison with the CE behavior of PSA standard that had been submitted to the same treatment (Fig. 3D). An additional clean-up step with water in another centrifugal filter markedly improved the pattern of serum fPSA (Fig. 3B) and increased migration velocity (tEOF = 14.38 min). A third clean® up treatment in a third Microcon YM-10 device led to a clean pattern of PSA isoforms from serum (Fig. 3C) with migration (tEOF = 13.90 min) more similar to that of PSA standard analyzed on the same day (tEOF = 13.35 min). As seen in Fig. 3, the use of effective electrophoretic migration (eff ) instead of migration time (tm ) as the migration parameter made it possible to compare the electropherograms directly. In addition, as expected, better repeatability was achieved by expressing migration as eff (RSD < 1.4% even for the smallest peaks) rather than as tm (Supplementary information, Table S2). The SDS-PAGE and subsequent WB analysis of this purified PSA from the serum sample showed that treatment with ethanolamine effectively dissociated most of the complexed-PSA (Fig. 4, lane 3). According to Peter et al. [15], the low percentage of non-dissociated PSA could be due to incomplete cleavage of a low amount of PSAACT complex or to other PSA-serpin complexes present in serum at low concentration. The molecular size of the non-cleaved PSA observed by Western blot (Fig. 4, lane 3, band 2) could correspond to PSA-ACT as described recently [24] and not to the complex of PSA with alpha 2-macroglobulin, which would be larger. The major
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Fig. 4. PSA detection of serum and immunopurified PSA from the same serum in a 12% SDS-PAGE under reducing conditions, followed by Western blot with anti-PSA antibodies. Lane 1: molecular weight markers. Lane 2: 0.5 L serum, which contains 10 ng of total PSA. Lane 3: 25 ng of immunopurified PSA from serum. Lane 4: 100 ng of PSA standard. Band 1 corresponds to free PSA and band 2 to the ACT-PSA complex; nicked forms are also indicated.
band of immunopurified PSA corresponding to fPSA was observed at about 35 kDa, and bands of lower molecular weight corresponding to nicked forms were not found in the serum (Fig. 4, lane 2) or in PSA purified from it (Fig. 4, lane 3). However, some nicked forms were detected in PSA standard in a relatively lower proportion than the mature PSA (Fig. 4, lane 4). 2-DE followed by WB analysis showed that the sample treatment methodology including dissociation of complexed PSA and immunopurification did not modify the pattern of fPSA obtained from serum (Fig. 5A and B). The 2-DE pattern of the serum PSA before and after immunopurification showed the same proportion of the main F1-F4 subforms at around 33 kDa (Fig. 5C). Nicked forms were very minor and hardly detected, in agreement with the results of the SDS-PAGE analysis of serum PSA mentioned above (Fig. 4). The conditions of 2-DE used for PSA analysis do not show the PSA complexed to ACT from the serum sample that focuses at higher molecular weight and at more acid pH as described previously [25]. fPSA 2-DE pattern is sensitive to PSA glycosylation changes [12] and to other post-translational modifications that could lead to variations in PSA pI and/or molecular weight. Therefore, the dissociation and immunopurification methodology described here allowed purifying the same PSA 2-DE subforms found in the original serum PSA, without selecting or selectively enriching any of them. The different CZE patterns of serum and seminal plasma PSA, both submitted to the sample treatment methodology developed, could be due to several reasons. These differences could reflect changes in the peptidic chain linkages (nicked forms), changes in glycosylation or in other types of modifications (phosphorylation, sulfation, etc) as a result of cancer. It has to be taken into account that the serum PSA was obtained from a patient with very advanced prostate cancer, while the commercial PSA standard was prepared from the seminal plasma of healthy donors. Regarding glycosylation, differences between the CZE profiles of serum PSA (Fig. 3C) and the PSA standard from seminal plasma (Fig. 2A) could be related to the differences in fucosylation and N-acetyl galactosamine content as well as in the linkage of sialic acid between PSA from a
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Fig. 5. 2-DE and Western blot analysis of PSA. (A) 2-DE pattern of 1 uL of serum of a prostate cancer patient, containing 20 ng of PSA, (B) 2-DE pattern of 15 ng of PSA purified from the same serum of a prostate cancer patient, (C) densitometry profile of the F1-F4 PSA spots.
cancer patient’s serum and from a control seminal plasma previously found using different analytical techniques [11,23,26]. Also it is possible that these differences in CZE patterns between samples arise from the fact that seminal plasma PSA is present only as fPSA, while the PSA purified from serum contains the original fPSA plus that released from the ACT-PSA complex. 4. Concluding remarks A method that makes it possible to purify PSA from serum in a way that is compatible with analysis of isoforms of the glycoprotein by CZE was developed. The sample preparation methodology necessary to analyze fPSA in serum samples consisting of treatment with ethanolamine and immunochromatographic purification does not notably alter the CD spectrum or the CE-UV profile of PSA. In addition, the 2-DE pattern of serum PSA is not modified by the sample treatment. Thorough removal of the reagents used in the sample preparation process is mandatory prior to the CE analysis for avoiding disturbances. Under the optimized sample preparation conditions, highly repetitive CD and CZE results are obtained. The CZE pattern of PSA from a serum sample of a prostate cancer patient show differences in comparison to the pattern of PSA standard from seminal plasma of healthy donors. These differences, that may reflect changes in glycosylation or in other features of the molecule, could arise either from the clinical stage of the donors or from the nature of the fluid. This analysis methodology, which includes sample preparation and capillary electrophoresis separation of PSA isoforms, makes it feasible to analyze the isoform pattern of this glycoprotein in rich PSA serum samples. In the future, CZE patterns of serum PSA from individuals with different clinical conditions (healthy, prostate cancer patients with different Gleason scores, and patients with non-malignant prostate diseases) could be compared to evaluate the usefulness of the CZE pattern as a prostate cancer marker.
For samples with lower PSA concentration, CE detection methods with enhanced sensitivity that maintain the separation of isoforms will be required. Work in this direction is currently being carried out in our laboratory. Acknowledgements The generous gift of the serum sample by the cancer patient donor is highly appreciated. We would also like to acknowledge the financial support from the Spanish Ministry of Economy and Competitiveness (MINECO, projects CTQ2013-43236-R, BIO201016922, and BIO2015-66356-R) and from the Generalitat de Catalunya, Spain (grant 2014 SGR 229). N. F.-G. and M. G. acknowledge the Ph.D. JAE-pre grant and the JAE-doc grant, respectively, from CSIC co-financed by the European Social Fund. D.N-C. acknowledges the contract in the frame of the Youth Guarantee Implementation Plans financed by the European Social Fund (ESF) and the Youth Employment Initiative (YEI). J.E. G.-L. acknowledges support by Award Number R01GM089759 from the National Institute of General Medical Sciences (NIH, U.S.A.). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jpba.2016.11.045. References [1] L.A. Torre, F. Bray, R.L. Siegel, J. Ferlay, J. Lortet-Tieulent, A. Jemal, Global cancer statistics, 2012, CA—Cancer J. Clin. 65 (2) (2015) 87–108. [2] R.L. Siegel, K.D. Miller, A. Jemal, Cancer statistics, 2015, CA—Cancer J. Clin. 65 (1) (2015) 5–29. [3] E.H. Kim, G.L. Andriole, Prostate-specific antigen-based screening: controversy and guidelines, BMC Med. 13 (2015). [4] J. Cuzick, M.A. Thorat, G. Andriole, O.W. Brawley, P.H. Brown, Z. Culig, R.A. Eeles, L.G. Ford, F.C. Hamdy, L. Holmberg, D. Ilic, T.J. Key, C. La Vecchia, H. Lilja, M. Marberger, F.L. Meyskens, L.M. Minasian, C. Parker, H.L. Parnes, S. Perner, H. Rittenhouse, J. Schalken, H.-P. Schmid, B.J. Schmitz-Draeger, F.H. Schroder,
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