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Capillary gel electrophoresis (CGE) has been widely used for analysis of proteins according to their size. However, to our knowledge, this technique has not been optimized to immunoglobulin A (IgA) analysis, a protein of current and emerging high interest in several fields. IgA is the first barrier of human body against pathogens. This protein in human milk and colostrum is essential for immune protection of newborns and treatment of milk for storage in Human Milk Banks may alter IgA. The emerging use of IgA as therapeutic treatment also encourages the development of analysis methods for this class of immunoglobulins. IgA is far more heterogeneously glycosylated and complex than the well-studied IgG molecules. IgA in serum is mainly monomeric (mIgA) with about 160 kDa, while in secretions such as saliva, milk, colostrum, etc, secretory immunoglobulin A (sIgA) is the predominant form. This is a dimer where both monomers are linked by the J-chain and the secretory component accounting all together for a MW higher than 400 kDa including the glycans. This size is far from the 225 kDa MW for which commercial CGE kits are intended. The general rules governing CGE behavior of analytes cannot be directly applied to every protein. Addressing studies directed specifically to target proteins is specially needed for the large size and highly complex target analytes of this study. In this work the effect of several factors on CGE analysis of human serum and colostrum IgA is studied. The feasibility of performing analysis of both IgA classes using a commercial CGE kit is shown. In addition, this work introduces another novelty by preparing tailor-made reproducible gel buffers and to characterize them in terms of dynamic viscosity, conductivity, and electroosmotic flow mobility in bare fused silica capillaries. The possibility of analyzing mIgA and sIgA in less than 10 min using these tailor-made gels is demonstrated. Inter-day variation (RSD) for the main peak of sIgA is 0.25% for migration time (tm) and 0.27% for percentage corrected peak area (Acorr)., This work is part of the I+D+I project PID2019-106405GB-I00 financed by MCIN/ AEI/10.13039/501100011033 and of project S2018/BAA-4393, AVANSECAL-II-CM financed by the Comunidad of Madrid and European funding from FSE and FEDER programs. P.T.- M. and L.G.-R. acknowledge contracts in the frame of the YGI Plans financed by the ESF and the YEI.

Glycoproteins are highly complex molecules. Knowledge of their heterogeneities in glycosylation and in other posttranslational modifications provides relevant information and requires high-resolution separation techniques. This chapter focuses on the capillary electrophoresis (CE) analysis of intact glycoproteins and glycopeptides and their fragments. It covers works published since 2015. The aim is to give a general vision of different strategies, approaches, and CE methods used to analyze these biomolecules. Their advantages and limitations are considered. Due to the high interest on monoclonal antibodies, a separated section is devoted to these immunoglobulins. Different detection systems are considered, and a separate section is dedicated to the coupling of CE with mass spectrometry in consideration to the additional information provided and to the large number of papers devoted to this coupling. As a final section, examples of applications of CE analysis of glycoproteins in the areas of biomarkers and therapeutic drugs are included.

Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.microc.2017.04.040, Isotachophoresis (ITP) coupled to zone electrophoresis (ZE), either free zone electrophoresis (FZE) or gel electrophoresis (GE), carried out mainly in capillaries and, although less frequently, also in microchips, is a powerful preconcentration and separation technique which has been successfully used for the study of many low molecular-weight analytes. However, this analytical technique has been scarcely applied for proteins separation. In this work, an on-chip transient ITP coupled to free zone electrophoresis (t-ITP-MFZE) mode with LIF detection is developed for the preconcentration and separation of the proteins α-lactalbumin and β-lactoglobulin. Firstly, for LIF detection, the proteins were off-chip fluorescently labeled with the fluorogenic reagent Chromeo P503. Then, several separation parameters in t-ITP-MFZE mode such as leading electrolyte, terminating electrolyte, separation voltage, and injection time were optimized to achieve the maximum sensitivity while maintaining an adequate resolution between α-lactalbumin and β-lactoglobulin in a single column configuration t-ITP. Using the optimized electrolytes (50 mM imidazole/HCl pH = 8 as leading electrolyte and 100 mM imidazole/12 mM HEPES pH = 8 as terminating electrolyte) separation of both proteins was achieved in less than 4 min with peak resolution of 1.5. The LODs were 55 nM and 380 nM, for α-lactalbumin and β-lactoglobulin, respectively, which are adequate for some food allergenicity studies. Finally, comparison of the optimized t-ITP-MFZE method to the equivalent MFZE method, carried out also in microchips but without the isotachophoretic preconcentration step, provided preconcentration indexes for both proteins around 10., The authors acknowledge the SpanishMinistry of Economy, Industry and Competitiveness (MINECO CTQ2013-43236-R) and CSIC (joint project 2009JP003 with the Japanese Society for Promotion of Science) for financial support. A.G.C. acknowledges CSIC for a JAE-Doc contract. This contract was co-financed by the European Union under the European Social Fund (ESF).

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., AcknowledgementsThe generous gift of the serum sample by the cancer patientdonor is highly appreciated. We would also like to acknowledgethe financial support from the Spanish Ministry of Economy andCompetitiveness (MINECO, projects CTQ2013-43236-R, BIO2010-16922, and BIO2015-66356-R) and from the Generalitat deCatalunya, Spain (grant 2014 SGR 229). N. F.-G. and M. G.acknowledge the Ph.D. JAE-pre grant and the JAE-doc grant, respec-tively, from CSIC co-financed by the European Social Fund. D.N-C.acknowledges the contract in the frame of the Youth GuaranteeImplementation Plans financed by the European Social Fund (ESF)and the Youth Employment Initiative (YEI). J.E. G.-L. acknowledgessupport by Award Number R01GM089759 from the National Insti-tute of General Medical Sciences (NIH, U.S.A.).

Serum levels of Prostate-Specific Antigen (PSA) are not fully specific for prostate cancer (PCa) diagnosis and several efforts are focused on searching to improve PCa markers through the study of PSA subforms that could be cancer associated. We have previously reported by 2DE a decrease in the sialic acid content of PSA from PCa compared to benign prostatic hyperplasia patients based on the different proportion of the PSA spots. However, faster and more quantitative techniques, easier to automate than 2DE, are desirable. In this study, we examined the potential of CE for resolving PSA subforms in different samples and compared the results with those obtained by 2DE. We first fractionated by OFFGEL the subforms of PSA from seminal plasma according to their pIs and analyzed each separated fraction by 2DE and CE. We also analyzed PSA and high pI PSA, both from seminal plasma, and PSA from urine of a PCa patient. These samples with different PSA spots proportions by 2DE, due to different posttranslational modifications, also presented different CE profiles. This study shows that CE is a useful and complementary technique to 2DE for analyzing samples with different PSA subforms, which is of high clinical interest.

Glycoproteins expressed in the human body can experience modifications as result of pathological situations. Detection of those changes can be useful as disease biomarkers. As a result of these modifications, size and/or electrical charge of the glycoprotein can be altered. Migration in capillary zone electrophoresis (CZE) is governed by the size to charge ratio of the analyte and therefore this separation technique can be used to monitor those modifications. At its turn, the alteration of the electrophoretical pattern of a given glycoprotein could be used as disease biomarker. To this aim, high repeatability for separation of a large number of peaks for a given glycoprotein is desirable.For prostate cancer, new markers are needed to decrease the high number of false positive results provided by the biomarkers currently used in clinics. In this sense, CZE methods for analysis of the several prostate specific antigen (PSA) peaks which this glycoprotein exhibit, called isoforms and containing one or more glycoforms, could be useful to study the PSA pattern as prostate cancer marker.In this study two complementary strategies to achieve both lot-to-lot capillary repeatability and high resolution of a large number of PSA isoforms are developed. Better performance and precision have been obtained for capillaries conditioned with HCl than for those conditioned with NaOH. Optimization of the background electrolyte (BGE) pH value to 8.0 and inclusion of 3 M urea on its composition were the two factors of highest impact for enhancing resolution of the highest number of PSA peaks. Under the optimized conditions for capillary conditioning and BGE pH and composition, long-term resolution of 10 isoforms of PSA was achieved. Inter-day (n = 3) %RSD was 0.55 for the ratio t/t, 1.15 for μ, and 5.02 for % A of the PSA peaks., AcknowledgementsFinancial support from the Spanish Ministry of Economy and Competitiveness (project CTQ2013-43236-R) is acknowledged.Noemi Farina-Gomez and Monica Gonzalez acknowledge the Ph.D.JAE-pre grant and the JAE-doc grant, respectively, from CSIC co-financed by the European Social Fund.

The Capillary Electrophoresis (CE) profile of isoforms (peaks) of a glycoprotein can be useful to show alterations in its posttranslational modifications (PTMs) linked to diseases. These changes can modify the electrophoretic mobility of these isoforms in a minor extent and, therefore, very reproducible CE methods are needed to detect them. In this chapter, a method for the analysis of prostate-specific antigen (PSA) by Capillary Zone Electrophoresis (CZE) with UV detection is detailed. High reproducibility in the separation of a large number of PSA isoforms is achieved by performing capillary conditioning in acid media and by using a background electrolyte (BGE) at pH 8.0 formulated with decamethonium bromide and urea.

In the last few years, biopharmaceuticals-therapeutic drugs which are generally obtained by using molecular biology techniques-have become a major growing sector in pharmaceutical industry. A large part of these biopharmaceuticals are therapeutic glycoproteins. The production of these drugs and their purification process are implying the development of efficient analytical methods, which allow quick and reliable control of the manufacturing process and ensuring the regulatory compliance about the quality of these drugs. Capillary gel electrophoresis (CGE) in the presence of sodium dodecyl sulfate (SDS) is becoming a method of choice in the quality control of these biopharmaceuticals. On the other hand, CGE can be improved if analyses are carried out in microchip format.This chapter reports a detailed microchips gel electrophoresis (MGE) method to separate glycosylated and deglycosylated forms of α1-acid glycoprotein (AGP) labeled with Chromeo P540, using SU-8 microchips and laser induced fluorescence detection. Due to the analogy between AGP and some therapeutic glycoproteins, we have selected AGP as a model system to illustrate the potential of MGE in the analysis of this type of biopharmaceutical compounds.

The Capillary Electrophoresis (CE) profile of isoforms (peaks) of a glycoprotein can be useful to show alterations in its post-translational modifications (PTMs) linked to diseases. These changes can modify the electrophoretic mobility of these isoforms in a minor extent and, therefore, very reproducible CE methods are needed to detect them. In this chapter, a method for the analysis of prostate specific antigen (PSA) by Capillary Zone Electrophoresis (CZE) with UV detection is detailed. High reproducibility in the separation of a large number of PSA isoforms is achieved by performing capillary conditioning in acid media and by using a background electrolyte (BGE) at pH 8.0 formulated with decamethonium bromide and urea.

In the last few years, biopharmaceuticals-therapeutic drugs which are generally obtained by using molecular biology techniques-have become a major growing sector in pharmaceutical industry. A large part of these biopharmaceuticals are therapeutic glycoproteins. The production of these drugs and their purification process are implying the development of efficient analytical methods, which allow quick and reliable control of the manufacturing process and ensuring the regulatory compliance about the quality of these drugs. Capillary gel electrophoresis (CGE) in the presence of sodium dodecyl sulfate (SDS) is becoming a method of choice in the quality control of these biopharmaceuticals. On the other hand, CGE can be improved if analyses are carried out in microchip format.This chapter reports a detailed microchips gel electrophoresis (MGE) method to separate glycosylated and deglycosylated forms of α1-acid glycoprotein (AGP) labeled with Chromeo P540, using SU-8 microchips and laser induced fluorescence detection. Due to the analogy between AGP and some therapeutic glycoproteins, we have selected AGP as a model system to illustrate the potential of MGE in the analysis of this type of biopharmaceutical compounds.

Pancreatic cancer (PDAC) lacks reliable diagnostic biomarkers and the search for new biomarkers represents an important challenge. Previous results looking at a small cohort of patients showed an increase in α-1-acid glycoprotein (AGP) fucosylation in advanced PDAC using N-glycan sequencing. Here, we have analysed AGP glycoforms in a larger cohort using several analytical techniques including mass spectrometry (MS), capillary zone electrophoresis (CZE) and enzyme-linked lectin assays (ELLAs) for determining AGP glycoforms which could be PDAC associated. AGP from 31 serum samples, including healthy controls (HC), chronic pancreatitis (ChrP) and PDAC patients, was purified by immunoaffinity chromatography. Stable isotope labelling of AGP released N-glycans and their analysis by zwitterionic hydrophilic interaction capillary liquid chromatography electrospray MS (μZIC-HILIC-ESI-MS) showed an increase in AGP fucosylated glycoforms in PDAC compared to ChrP and HC. By CZE-UV analysis, relative concentrations of some of the AGP isoforms were found significantly different compared to those in PDAC and HC. Finally, ELLAs using Aleuria aurantia lectin displayed a significant increase in AGP fucosylation, before and after AGP neuraminidase treatment, in advanced PDAC compared to ChrP and HC, respectively. Altogether, these results indicate that α1-3 fucosylated glycoforms of AGP are increased in PDAC and could be potentially regarded as a PDAC biomarker. M.B. acknowledges the University of Girona for a pre-doctoral fellowship. This work was supported by the Government of Catalonia (grant 2014 SGR 229), the Spanish Ministry of Science and Innovation (grant BIO 2010-16922, awarded to R. P) and the Spanish Ministry of Economy and Competitiveness (grant CTQ2013-43236, awarded to M.F. and grant CTQ2011-27130, awarded to V. S-N).

Serum levels of Prostate-Specific Antigen (PSA) are not fully specific for prostate cancer (PCa) diagnosis and several efforts are focused on searching to improve PCa markers through the study of PSA subforms that could be cancer associated. We have previously reported by 2DE a decrease in the sialic acid content of PSA from PCa compared to benign prostatic hyperplasia patients based on the different proportion of the PSA spots. However, faster and more quantitative techniques, easier to automate than 2DE, are desirable. In this study, we examined the potential of CE for resolving PSA subforms in different samples and compared the results with those obtained by 2DE. We first fractionated by OFFGEL the subforms of PSA from seminal plasma according to their pIs and analyzed each separated fraction by 2DE and CE. We also analyzed PSA and high pI PSA, both from seminal plasma, and PSA from urine of a PCa patient. These samples with different PSA spots proportions by 2DE, due to different posttranslational modifications, also presented different CE profiles. This study shows that CE is a useful and complementary technique to 2DE for analyzing samples with different PSA subforms, which is of high clinical interest.

The analysis of glycoprotein isoforms is of high interest in the biomedical field and clinical chemistry. Many studies have demonstrated that some glycoprotein isoforms could serve as biomarkers for several major diseases, such as cancers and vascular diseases, among others. Capillary zone electrophoresis (CZE) is a well-established technique to separate glycoprotein isoforms, however, it suffers from limited sensitivity when UV-Vis detection is used. On the other hand, with laser-induced fluorescence (LIF) detection, derivatization reaction to render the proteins fluorescent can destroy the resolution of the isoforms. In this work, a derivatization procedure through the thiol groups of glycoproteins using either 5-(iodoacetamide) fluorescein (5-IAF) or BODIPY iodoacetamide is presented with the model protein of alpha-1-acid glycoprotein (AGP). The derivatization process presented enabled high-resolution analysis of AGP isoforms by CZE-LIF. The derivatization procedure was successfully applied to label AGP from samples of serum and secretome of artery tissue, enabling the separation of the AGP isoforms by CE-LIF in natural samples at different concentration levels.

a-1-Acid glycoprotein (AGP) is a glycoprotein that presents different forms in the same in- dividual, depending on the amino acid sequence and/or on the carbohydrate distribution of each form. Changes in these two types of heterogeneities are related to pathophysiological states. The aim of this work is to study the possibility of comparing AGP samples in terms of their CIEF profiles, what would facilitate in a future to perform studies about the role of AGP as a disease marker. In the present study, the CIEF profiles of AGP samples purified from sera of healthy donors and of ovary cancer and lymphoma patients are qualitatively and quantitatively compared. To make possible the comparison of those electrophoretical profiles, reliable assignment of AGP peaks is necessary. A computer program developed in our laboratory is used to select the migration parameters that make possible an accurate assignment of AGP peaks. Percentages of correct assignment of AGP peaks using the migration time of each peak relative to the migration time of an internal standard close to 95% are achieved. After peak assignment, a different distribution of the area percentage of AGP forms is observed when comparing samples from diseased and healthy individuals, the most acidic AGP forms being present in a higher proportion in the samples from cancer patients. Although the number of samples studied is too low to get any clinical significance from these results, this work provides a way to study the role of AGP as a biomarker.

Human serum albumin (HSA) is added to some pharmaceutical preparations as an excipient. This is the case for some of the commercial preparations of recombinant erythropoietin (rEPO). Differences in the number of the sialic acid moieties in the different rEPO glycoforms confer to these forms different net charges and different bioactivity. Knowledge of the isoforms present in each pharmaceutical product is then of interest. Differences in net charge of the rEPO forms make possible their separation by electrophoretical methods. However it has been observed in our laboratory that the amount of HSA usually present in these drug formulations interferes or even precludes separation of rEPO bands by capillary zone electrophoresis (CZE). In this work, an immunochromatographic method to remove HSA from rEPO biopharmaceutical formulations and a procedure to concentrate the sample that is needed to be performed prior to the analysis by CZE are developed. A home-made computer program to compare the percentage of correct assignments of electrophoretical bands provided by different migration parameters is used to study the effect of HSA remaining in samples on the accuracy of assignment of rEPO bands. When there exists a residual concentration of HSA in the sample (, The authors are grateful to foundation R. Areces and to Spanish Ministry of Science and Technology (project TIC2003- 01906) for providing financial support, to European Pharma- copoeia for providing the rEPO BRP used as standard sample, to Dr. Michel Girard (Health Canada) for the gift of epoetin alfa;, and to Applied Biosystems for the immunocolumn kindly donated. P. L-Q. and I. L. acknowledge Spanish Ministry of Education and Science and C. S. I. C., respectively, for fellowships.

9 pages, 6 figures.-- PMID: 15495411 [PubMed].-- Printed version published Sep 2001., Part of this work is included in patents (ES2167238 and WO0190755)., The retention of organochlorinated compounds on an immunochromatographic column is studied. The compounds considered are usually found together in real samples of environmental concern, and include chlorinated biphenyls, chlorinated dibenzo-p-dioxins, chlorinated dibenzofurans, and organochlorinated insecticides. The different retention observed for different compounds is interpreted in light of the structural similarities of the compound studied with that used as a hapten to raise the antibodies employed as ligands in the immunochromatographic column. Differences in retention of the organochlorinated compounds on the immunocolumn make it possible to fractionate them. Mixtures of phosphate buffer solution and ACN in different ratios were used as desorption agents. Depending on the percentage of ACN employed, different fractionations of the organochlorinated compounds are obtained. The use of 20% ACN allows fractionation of most insecticides from chlorinated biphenyls, dioxins, and furans. Besides, chlorinated biphenyls of different chlorination degree can be separated. Moreover, pentachlorinated dioxins and pentachlorinated furans are separated from hexachlorinated biphenyls. Fractionation is of critical importance from a practical point of view, as it avoids some of the interferences that otherwise take place during the subsequent gas chromatography/mass spectrometry (GC/MS) analysis of these compounds. In this way, immunochromatography makes it possible to perform in a single step all the processes (extraction, clean-up, concentration, and fractionation) that are needed prior to the GC/MS analysis of these analytes in aqueous samples. An additional advantage of this method is the reusability of the immunoaffinity chromatography column for more than 500 times., This work has been supported by the Spanish Ministry of Science and Technology (Projects INIA CAL00-041 and BQU2001-2270) and by the Spanish Ministry of Education and Culture (Project 2FD97-0621).

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was used to detect an immune complex formed between beta-lactoglobulin and polyclonal anti-beta-lactoglobulin antibody in the gas phase. The most important experimental parameters to detect such a specific antibody-antigen complex by MALDI were the use of solutions at near-neutral pH and of sinapinic acid matrix prepared by the dried-droplet method. Under such conditions, predominantly one but also two molecules of antigen protein were complexed by the antibody. Specific formation of the antibody-antigen complex was confirmed by performing competitive reactions. Addition of antibody to a 1:1 mixture of beta-lactoglobulin and one control protein resulted not only in the appearance of the expected antibody-antigen complex, but also in a strong decrease in the free beta-lactoglobulin signal, while the abundance of the control protein was not influenced.

Human erythropoietin (hEPO), an endogenously produced glycoprotein, plays a fun- damental role in erythropoiesis controlling the formation of red blood cells. Production of recombinant erythropoietin (rEPO) by molecular biology techniques has made pos- sible its therapeutic use together with its abuse in competitive sports. Since the glyco- sylation pattern of rEPO depends, among other things, of the cell line used for pro- duction, it presents a glycoform scheme different from manufacturer to manufacturer and different also from endogenous hEPO. The separation of these different glyco- forms is interesting since it could be useful: i) for determining the glycoform distribu- tion in recombinant products from different batches and/or manufacturers, ii) for studying the link between glycosylated forms and their activity, and iii) for helping to discriminate between endogenous and recombinant EPO. This paper desribes the development of three different Capillary Electrophoresis methods that use UV detec- tion to separate rEPO glycoforms. The special features of the three methods are dis- cussed in terms of resolution of bands of rEPO glycoforms, method reproducibility, and compatibility with other more sensitive detection systems such as laser induced fluorescence using on-column derivatization protocols or more selective ones such as mass-spectrometry., The authors are grateful to European Pharmacopoeia for providing rEPO BRP used as sample in this work and to M.T. Veledo for carrying out the CE-LIF experiments. This work was supported by an International Olympic Commit- tee project (1767/2000/HMS/sls) and by a CICYT project (AGL2000-1480).

Uridine diphosphate galacturonate 4-epimerases Introduction (UDPGLEs) are enzymes that convert UDP-glucuronate into UDP-galacturonate. Although the presence of UDPGLEs hasbeen reported in prokaryotic and eukary-otic organisms, the genes coding for these enzymes are completely unknown. The galacturonic acid-con-taining capsular polysaccharide of Streptococcus pneumoniae type 1 is synthesized through the action of a specific UDPGLE. We have constructed a defined deletion mutant in the cap1J gene (one of the 15 cap1 genes responsible for the synthesis of the type 1 cap-sule) that exhibited an unencapsulated phenotype. This mutant was unable to synthesize UDPGLE, sug-gesting that Cap1J was the type 1-specific UDPGLE of S. pneumoniae. Escherichia coli cells harbouring the recombinant plasmid pRMM38 (cap1J ) overpro-duced a 40 kDa protein, characterized as Cap1J on the basis of the N-terminal amino acid sequence analysis, and expressed high levels of enzymatically active Cap1J epimerase. Cap1J was partially purified, although puri-fication to electrophoretic homogeneity inactivated the enzyme irreversibly. The enzyme has the following characteristics: Km for UDP-glucuronate, 0.24 mM; pH optimum, 7.5; equilibrium constant (in the direction of UDP-galacturonate formation), 1.3; and an approximate Mr of 80 000 for the active form. The Cap1J protein exhibited a fluorescence emission spectrum similar to that of NADH. Upon inactivation with p-hydroxymer-curibenzoate, the addition of NADþ and 2-mercap-toethanol were sufficient to reactivate the enzyme. In an early work, Smith et al. (1957) first reported the isolation of UDP-galacturonic acid (UDP-GalA) from a type 1 Streptococcus pneumoniae isolate. Additional studies showed that cell-free extracts prepared from type 1 pneumococci contained an enzymatic activity capable of converting UDP-glucuronic acid (UDP-GlcA) into UDP-GalA (Smith et al., 1958; 1960). UDP-GalA serves as a pre-cursor for the synthesis of type 1 pneumococcal capsular polysaccharide, as demonstrated by the finding that mutants deficient in UDP-glucuronate 4-epimerase (UDP-GLE) (EC 5.1.3.6) activity were unencapsulated (Austrian et al., 1959). Several independent reports have also detec-ted the presence of UDPGLE in the cyanobacterium Ana-baena flos-aquae (Ankel and Tischer, 1969; Gaunt et al., 1972; 1974; Maitra et al., 1974) and in different plants (Neufeld et al., 1958; Feingold et al., 1960; Neufeld, 1966; Dalessandro and Northcote, 1977). Unfortunately, none of these enzymes have been purified to sufficient homo-geneity, and attempts to clone the corresponding genes have not been documented, hampering a reliable charac-terization of this type of protein. We have reported recently the characterization of the gene cluster responsible for capsular polysaccharide bio-synthesis in type 1 strains of S. pneumoniae (Garcı´a et al., 1997; Mun˜oz et al., 1997) and identified the gene cap1K as that coding for the UDP-glucose (UDP-Glc) dehydrogenase responsible for the synthesis of UDP-GlcA from UDP-Glc. We report here experimental evidence, We thank J. C. Díez Masa for his invaluable help with the chromatographic separation of UDP-glucuronic acid and UDP- galacturonic acid, F. J. Medrano for his advice with the spectro- fluorometer, P. García and J. L. García for critical reading of the manuscript, and E. Cano, M. Carrasco and V. Muñoz for tech- nical assistance. This work was supported by grant PB96-0809 from the Dirección General de Investigación Científica y Téc- nica. R. Muñoz was a recipient of a Contrato Temporal de Investigadores from the CSIC.

Elution of a commercial sample of bovine serum albumin (BSA) in a reversed-phase (RP) HPLC system at room temperature gives a distorted peak. If a shallow gradient is used during elution a split peak is observed. The nature of the several parts of this multiple peak is studied using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), size-exclusion chromatography (SEC), amino acid analysis, re-elution in RP-HPLC of collected fractions, capillary electrophoresis (CE), and matrix assisted laser desorption ionization-mass spectrometry (MALDI-MS). This study demonstrates that the split peak of BSA observed in these chromatographic conditions is due to the monomer, dimer and other aggregates existing in the commercial sample of the BSA used. Moreover, it is proved that typical RP-chromatographic conditions do not cause aggregation of BSA., The authors thank F. Soriano for his skillful technical assistance. This work was supported by CICYT (project ALI 97 – 0630).

The chromatographic behavior of beta-lactoglobulins when eluted in hydrophobic interaction chromatography systems is studied. By modifying some factors, such as pH and temperature, the relationship between shape of the chromatographic peak and protein structure is shown. At pH 4.5 and low temperature multiple peaks for beta-LG A and beta-LG B are observed and assigned to aggregates. The effects of other parameters, besides pH and temperature, such as volume and concentration of injected sample, contact time between protein and stationary and/or mobile phases, and nature and concentration of mobile phase upon aggregation are studied. Comparison of the chromatographic behavior of both variants of beta-lactoglobulin is made.

Sample preparation and laser-induced fluorescence detection are two key steps of the analytical methodology to determine by capillary electrophoresis low concentrations of proteins in complex sample matrices. In this chapter the options of performing both steps in different ways are shown by detailing the analysis of the allergen β-lactoglobulin in food products for infants and the analysis of the isoforms of alpha 1-acid glycoprotein, a potential biomarker, in serum and secretome.

This chapter describes a complete procedure for obtaining protein fingerprints of microorganisms using capillary electrophoresis (CE) with laser-induced fluorescence detection (LIF). Staphylococcus aureus, a human pathogen responsible of frequent and resistant infections, is used as model microorganism to show the feasibility of this procedure. Bacteria are grown in different culture media or submitted to temperature or nitrosative stress conditions. After the growth of the bacteria, the protein extracts are obtained by cell lysis using sonication. The water-soluble fraction of these lysates is derivatized on-capillary with the fluorogenic reagent 3-(2-furoyl)quinoline-2-carboxaldehyde. The fluorescent products are analyzed by CE and detected by LIF. Practical advices for the interpretation of the electropherograms are given. To do so, the variations of the protein fingerprints of the bacteria with the culture conditions, such as growth medium, or the stressing conditions, such as heat shock or nitrosative stress, are used as example.

Variations in the amino acid sequence, glycosylation, and/or other posttranslational modifications in glycoproteins give rise to different molecules of the glycoprotein called forms. Qualitative and/or quantitative alterations in these forms are related to pathophysiological situations in the individuals. In this study, a methodology to analyze these differences in forms of the alpha 1-acid glycoprotein (AGP) between healthy individuals and patients with two different vascular diseases is detailed. The whole methodology includes a sample preparation method based on immunochromatography, a capillary electrophoresis method for separation of AGP peaks (isoforms), and statistical methods (Linear Discriminant Analysis) for sample classification. As a result, it is shown that the methodology proposed allows studying the role of AGP isoforms as potential vascular disease biomarkers., Financial support from Comunidad de Madrid (project S-GEN/0247/2006) and the Spanish Ministry of Science and Innovation (project CTQ2009-09399) is acknowledged. Angel Puerta acknowledges the Spanish National Research Council (CSIC) for a contract in the JAEdoc program.

Sample preparation and laser-induced fluorescence detection are two key steps of the analytical methodology to determine by capillary electrophoresis low concentrations of proteins in complex sample matrices. In this chapter the options of performing both steps in different ways are shown by detailing the analysis of the allergen β-lactoglobulin in food products for infants and the analysis of the isoforms of alpha 1-acid glycoprotein, a potential biomarker, in serum and secretome.

This chapter describes a complete procedure for obtaining protein fingerprints of microorganisms using capillary electrophoresis (CE) with laser-induced fluorescence detection (LIF). Staphylococcus aureus, a human pathogen responsible of frequent and resistant infections, is used as model microorganism to show the feasibility of this procedure. Bacteria are grown in different culture media or submitted to temperature or nitrosative stress conditions. After the growth of the bacteria, the protein extracts are obtained by cell lysis using sonication. The water-soluble fraction of these lysates is derivatized on-capillary with the fluorogenic reagent 3-(2-furoyl)quinoline-2-carboxaldehyde. The fluorescent products are analyzed by CE and detected by LIF. Practical advices for the interpretation of the electropherograms are given. To do so, the variations of the protein fingerprints of the bacteria with the culture conditions, such as growth medium, or the stressing conditions, such as heat shock or nitrosative stress, are used as example.

The test used in clinics as prostate cancer (PCa) biomarker, based on the concentration of the glycoprotein prostate-specific antigen (PSA) in serum, leads to an elevated number of false positives. In the search for new PCa biomarkers, analysis of the proportions of different groups of glycoforms of PSA is promising. Peaks of PSA, called isoforms and containing one or several glycoforms of the glycoprotein, can be separated by CE. For those samples in which PSA concentration is very low, a very sensitive detection technique, such as LIF, would be required. However, CE separation of fluorescently labeled isoforms of glycoproteins is challenging. In this work, three different methods of fluorescent derivatization of PSA were assayed with the aim of finding conditions allowing labeling of the glycoprotein compatible with CE resolution of its isoforms. NanoOrange, as a noncovalent label; 5-(iodoacetamide) fluorescein and BODIPY® FL C1 -IA, as covalent tags of thiol groups; and Chromeo™ P503, as a covalent tag of amino groups, were tried. Only the derivatization with the P503 fluorogenic dye led to the resolution by CE-LIF of several isoforms of labeled PSA. Adapting this derivatization method to be performed on-column leads to a reduction in labeling time from 4 h to 45 s. Automation of the whole analysis permitted to carry out fluorescent labeling and CE separation of PSA isoforms in less than 12 min.

An immunoaffinity purification method coupled on-line to capillary electrophoresis (IACE) which allows the determination of several isoforms of intact alpha-1 acid glycoprotein (AGP) in serum samples using UV detection is developed. The immunoaffinity step is based on anti-AGP antibodies (Abs) covalently bound to magnetic beads (MBs) which are captured at the inlet end of the capillary using permanent magnets placed inside the cartridge of the CE instrument. The on-line method includes injection of the MBs with the Ab bound (MBs-Ab) and their trapping by the magnets at the entrance of the separation column, injection of serum sample and capture of AGP by the Abs, release of captured AGP, focus of desorbed protein, separation of AGP isoforms, and removal of MBs-Ab. The optimization of the different factors involved in each step allowed purification, separation and detection of AGP isoforms in a single electrophoretic analysis in about 1. h. Automation, sample and reagents consumption as well as analysis time was improved compared to off-line alternatives which use purification of AGP in an immunochromatographic column and CE separation of AGP isoforms in two independent operations. The analytical methodology developed allows the separation of 10 AGP isoforms in serum samples from a healthy donor. For a serum sample, precision (expressed as relative standard deviation) in terms of corrected area percentage was better than 0.5% for each peak accounting for more than 10% of total AGP and it was better than 4.0% in terms of relative migration time of each AGP isoform considering the whole process. © 2013 Elsevier B.V.

The value in environmental analysis of methods based on combinations of antigenicity and chromatographic properties is shown. Immunochromatographic analysis is reviewed as applied to samples of environmental concern. Other coupling methods which are of potential interest, but have not yet been applied in environmental studies, are also considered. The superiority is shown of immunochromatographic analysis over high-performance liquid chromatography and classical immunoassays performed independently.

Variations in the amino acid sequence, glycosylation, and/or other posttranslational modifications in glycoproteins give rise to different molecules of the glycoprotein called forms. Qualitative and/or quantitative alterations in these forms are related to pathophysiological situations in the individuals. In this study, a methodology to analyze these differences in forms of the alpha 1-acid glycoprotein (AGP) between healthy individuals and patients with two different vascular diseases is detailed. The whole methodology includes a sample preparation method based on immunochromatography, a capillary electrophoresis method for separation of AGP peaks (isoforms), and statistical methods (Linear Discriminant Analysis) for sample classification. As a result, it is shown that the methodology proposed allows studying the role of AGP isoforms as potential vascular disease biomarkers.

The analysis of glycoprotein isoforms is of high interest in the biomedical field and clinical chemistry. Many studies have demonstrated that some glycoprotein isoforms could serve as biomarkers for several major diseases, such as cancers and vascular diseases, among others. Capillary zone electrophoresis (CZE) is a well-established technique to separate glycoprotein isoforms, however, it suffers from limited sensitivity when UV-Vis detection is used. On the other hand, with laser-induced fluorescence (LIF) detection, derivatization reaction to render the proteins fluorescent can destroy the resolution of the isoforms. In this work, a derivatization procedure through the thiol groups of glycoproteins using either 5-(iodoacetamide) fluorescein (5-IAF) or BODIPY iodoacetamide is presented with the model protein of alpha-1-acid glycoprotein (AGP). The derivatization process presented enabled high-resolution analysis of AGP isoforms by CZE-LIF. The derivatization procedure was successfully applied to label AGP from samples of serum and secretome of artery tissue, enabling the separation of the AGP isoforms by CE-LIF in natural samples at different concentration levels.

Human erythropoietin (hEPO), a hormone involved in the formation of red blood cells, is a 30. kDa glycoprotein with a high carbohydrate content. The production of recombinant hEPO has made possible its widespread therapeutic use and its banned use in competition sports. Methods to analyze EPO and other erythropoiesis stimulating agents (ESAs) are necessary for the characterization and quality control of these biopharmaceuticals and also for doping control. In this paper, high resolution separation methods, namely high performance liquid chromatography (HPLC) and capillary electrophoresis (CE), with special attention to CE-coupled mass spectrometry, are reviewed. The usefulness of these techniques when applied in different modes to separate the glycoprotein isoforms, aggregates or excipients are detailed. In addition, sample preparation methods that have been applied to ESA samples for subsequent determination by HPLC or CE, as well as the potential compatibility of other preparation methods, are discussed. Applications of the HPLC and CE methods regarding regulatory considerations for biopharmaceuticals analysis, with emphasis on biosimilars, and doping control are also included. Finally, limitations of the present methods and their possible solutions are considered. © 2011 Elsevier B.V.

Prostate-specific antigen (PSA) is the usual biomarker for prostate cancer (PCa). However, its lack of selectivity has lead to the search for new biomarkers. PSA glyco- sylation seems to depend on the pathophysiological conditions of the individual. Thus, methods to separate PSA isoforms (peaks) to study their role as PCa markers are needed. In this work, CE methods for PSA isoforms separation, based on the use of different dynamic coatings, are developed using UV detection. Three complementary CE methods allowing the separation of 8 or 9 PSA isoforms are selected. The longest method takes only 17 min, while the shortest one separates 9 isoforms in o 8 min. Depending on the isoforms of interest for their use as PCa biomarker, the CE method to be used can be chosen or various of them can be combined. A remarkable aspect of these methods is that the BGEs employed are devoid of compounds with primary amino groups, making the CE methods compatible with fluorescent on-column derivatization through amino residues. As a proof-of-concept, a preliminary result shows that LIF detection of labeled PSA analyzed by one of the three developed methods permits detection of glycoprotein isoforms.

7 páginas, 3 figuras, 2 tablas.-- et al., α-1-Acid glycoprotein (AGP) is a serum glycoprotein that presents several isoforms. Changes in the isoforms of AGP have been related to different pathological states including cardiovascular diseases (CVDs) such as acute myocardial infarction. However, to our knowledge, the role of variations of AGP isoforms as a potential biomarker of atherothrombosis has not been addressed. In this work, a preliminary study about differences in the capillary zone electrophoresis (CZE) profile of intact (non-hydrolyzed) AGP isoforms between healthy individuals and patients with atherothrombosis, specifically abdominal aortic aneurysm (AAA) and carotid atherosclerosis (CTA), has been performed. Biological samples (plasmas and sera) were analyzed by CZE after immunoaffinity chromatography purification. Up to 13 peaks corresponding to groups of isoforms of intact AGP from plasma samples were detected by CZE-UV. Electrophoretic profiles were aligned, peaks assigned, and linear discriminant analysis (LDA) of percentage of the corrected areas of AGP peaks was employed to discriminate and classify the CZE profiles of AGP samples. LDA enabled to accomplish 92.9% of correct classification of the AGP samples when the three groups of samples were considered. Besides, the LDA model showed high predictive power in the groups healthy vs. sick, healthy vs. AAA, and healthy vs. CTA. The described method was a successful approach to study the potential of AGP isoforms profile as a biomarker of atherothrombosis. To the best of our knowledge this has been the first time that a possible role of the CZE profile of intact AGP isoforms as a biomarker of vascular diseases has been demonstrated., Financial support from Comunidad de Madrid (project S-GEN/ 0247/2006), the Spanish Ministry of Science and Innovation (projects PCI2006-A7-0646 and CTQ2009-09399), and the European Union (FAD Project, FP7, Health F2-2008-206647) is acknowledged. Angel Puerta acknowledges the Spanish National Research Council (C.S.I.C.) for a contract in the JAEdoc program.

Capillary electrophoresis (CE) coupled with laser-induced fluorescence detection (LIF) has allowed to obtain protein fingerprints, which have demonstrated to be useful in microorganisms characterization. In this work, protein fingerprints of two species of Staphylococcus grown in different culture media and submitted to temperature and nitrosative stress were studied by CE-LIF. After the growth of the bacteria, protein extracts were obtained by cell lysis using sonication. The water-soluble fraction of these lysates was derivatized on-capillary with a fluorogenic dye, 3-(2-furoyl)quinoline-2-carboxaldehyde. The fluorescent products were analyzed by CE using phosphate buffer containing submicellar concentrations of sodium pentanesulfate and detected by LIF. Different protein fingerprints were obtained depending on the bacterial specie studied, indicating the usefulness of this method for the identification of different species of the same bacterial genus. It was also demonstrated that the CE protein fingerprints were dependent on the culture conditions, such as growth medium, or on stressing conditions, such as heat shock or nitrosative stress, Our acknowledgement to Spanish Ministry of Science and Innovation (PATSENS project, #PSE-01000-2008-6), to Comunidad de Madrid (S-GEN/0247/2006 project) and to Domingo Martínez Foundation for financial support

Dairy products can induce allergic reactions even when present at very low levels, such as levels found in involuntary contamination during food manufacturing. β-Lactoglobulin (βLG) is the main allergen in cow’s milk. The objective of this work was to develop a sensitive method for βLG detection in baby foods through the optimization of an innovative sample preparation method. Three types of baby foods deliberately contaminated with dairy products or dairy desserts were sterilized to simulate the potential contamination occurring during manufacturing and then used as samples. Different sample preparation methods were compared. The best results were provided by an extraction solution containing β-mercaptoethanol, guanidine hydrochloride, and a saline solution. An ELISA method was optimized for the detection of βLG (LOD = 9.7 10 -13 M). The developed method allowed detection of even 1 part of dairy product in 100,000 parts of baby food for some of the analyzed foods.

Differences in a-1-acid glycoprotein (AGP) peptidic and glycan moieties originate several isoforms, whose modifications have been related to different pathophysiological situa- tions. Differences in the isoforms of AGP existing in serum of individuals suffering from different diseases compared to healthy ones could be potentially used as biomarkers. CZE has been proven to be a useful technique for the analysis of glycoprotein isoforms. However, direct CZE analysis of AGP isoforms in serum samples needs efficient puri- fication methods that allow the protein analysis. In this work two new and fast methods to purify AGP from human serum are evaluated in regard to their effect on the deter- mination of isoforms of the intact glycoprotein by CZE-UV and by a developed CZE-ESI- TOF-MS method. Both preparation methods, which differ in the pre-treatment of the sample prior to an anti-AGP immunochromatographic step are shown to be adequate to analyze isoforms of intact AGP. Comparison of both purification methods by CZE-UV and CZE-ESI-TOF-MS indicates that serum AGP purified without acidic precipitation as pre-treatment is more adequate due to AGP higher yield, which leads to better CZE-Mass spectra. Both CZE methods show no indication that acidic precipitation influences the glycosylation (including sialylation) of AGP.

Alpha 1-acid glycoprotein (AGP) is a very heterogeneous glycoprotein presenting several isoforms due to variations in its polypeptidic and glycosidic moieties. Differences in AGP isoforms between healthy and diseased individuals have been related to different pathological situations such as cancer or car- diovascular diseases, among others. Capillary electrophoresis study of the role of AGP isoforms as biomarkers requires prior purification of AGP from biological samples. Current AGP purification methods are time-and labour-consuming, and generally they have not been proven to be compatible with capil- lary electrophoresis analysis. In this work, different methods for AGP purification from human serum are developed and compared. The applicability of acidic precipitation and immunoaffinity chromatographic methods for AGP purification are studied. Two different immunoaffinity approaches are employed; in the first one, interferents present in the AGP sample are captured and removed, and in the second one, AGP is retained in a house-made anti-AGP column, being in this way isolated from the rest of interfer- ents of the sample. Best results in AGP purification from human serum to be analyzed by capillary zone electrophoresis (CZE) were obtained when acidic purification was combined with immunoaffinity chro- matography (IAC) employing the house-made anti-AGP column. The method was shown not to alter the proportion of AGP peaks due to isoforms existing in AGP samples. The applicability of this fast and easy purification method developed for analyzing by CZE isoforms of AGP from natural serum samples by CZE is demonstrated., Sara Ongay and Izaskun Lacunza acknowledge CSIC for contracts to perform Ph.D. Theses. Drs. Enrique Caso and Maria del Val Toledo are acknowledged for providing the serum samples. Financial sup- port from the Spanish Ministry of Science and Innovation (projects CTQ 2006-05214, HH 2006-0013, and PSE-010000-2008-6) and Comunidad de Madrid (project S-GEN-0247-2006) is acknowledged.

Beta-Lactoglobulin (BLG) is the main allergenic protein in cow’s milk and can cause allergy even when present at very low concentration. The aim of this work is to develop an innovative sample prepara- tion method fully compatible with capillary electrophoresis and laser-induced fluorescence detection for improving the sensitivity when analyzing BLG. Different types of baby food were on purpose contaminated with diverse dairy desserts and submitted to thermal treatment to simulate potential contamination at production. Sample preparation prior to CE analysis was performed by the classical extraction method and by the innovative one, and the results were compared. Analysis was performed by capillary electrophoresis with laser-induced fluorescence detection. The innovative method permitted to detect contaminations as low as 1 part of yoghurt in 10 000 parts of baby food., Financial support from the Spanish Ministry of Education and Science (projects DEP2006-56207 C03-01 and CTQ2006-05214) is acknowledged.

The vascular endothelial growth factor 165 (VEGF165) is the predominant form of the complex VEGF family. This glycoprotein has, among others, an angiogenic effect in many physiological and pathological events. For this reason, its roles as a biomarker and as a therapeutic drug have been considered. However, very little is known about the existence of different forms of VEGF165 arising from glycosylation and other potential PTMs. This aspect is crucial because it is known that for other glycoproteins the ratio between these isoforms actually acts as a biomarker for certain diseases and other physiological states. In addition, for therapeutic use of glycoproteins it is known that the biological activity may differ for the various isoforms. In this work CE methods to separate up to seven peaks without baseline resolution containing various forms of VEGF165 are developed. Using a computer program previously developed in-house peak assignment could be performed with accuracy close to 100%. In this way, comparison between recombinant human VEGF165 expressed in insect cells, which is a glycosylating system, and in Escherichia coli cells, which are unable of performing glycosylation of proteins, has been possible. The methods developed, besides providing information about the existence of several forms of VEGF165, mean a starting point that permits the study of the role of VEGF165 as a potential biomarker of different diseases and physio- logical processes and to perform quality control of the recombinant drug during manufacturing. To the best of our knowledge this is the first time that CE methods for VEGF165 have been developed., S.O. acknowledges C.S.I.C. for a predoctoral fellowship. A.P. acknowledges the Spanish Ministry of Education and Science for a postdoctoral grant. Prof. J.B. holds a senior research position financed by the Swedish Research Council. The finan- cial support from the Spanish Ministry of Education and Science (project CTQ2006-05214), Fundacio´n Ramo´n Areces, Comu- nidad de Madrid (Project S-GEN/0247/2006), and the Swedish Research Council (projects 621-2005-5279, 629-2002-6821 J.B.) is acknowledged. The authors have declared no conflict of interest.

The vascular endothelial growth factor (VEGF) is involved in different sicknesses (cardiovascular diseases, cancer, and other). Out of the many components of the VEGF family, the A splice variant with 165 amino acids (VEGF165) is the main component. In spite of the potential as biomarker that this protein has, information about its physico-chemical characteristics is scarce. In this study CIEF and MALDI-TOF-MS methods for intact recombinant human VEGF165 are developed and applied to analyze this glycoprotein expressed in glycosylating (Sf 21 insect cells) and non-glyco- sylating (Escherichia coli) systems. Different parameters influencing the CIEF separation were studied. The developed CIEF method allowed for the separation of up to seven peaks in the VEGF165 expressed in insect cells and up to three in VEGF165 expressed in E. coli. The use of the presented method permits the estimation of the apparent pI of the different forms of VEGF165 expressed in insect cells to be in a range of 6.8–8.2. The three peaks with intermediate pI values are observed in the protein expressed in both systems, insect cells and E. coli. The MALDI-TOF-MS method enabled to a rapid partial char- acterization of VEGF165 based on its MS fingerprint. MALDI-MS analysis of VEGF165 expressed in insect cells shows the presence of, at least, four forms or groups of forms of VEGF165 as a result of the different PTMs of the protein. According to the MALDI-MS analysis, VEGF165 expressed in E. coli was produced as a very homogeneous protein, although the results suggest the existence of some PTMs in the protein. The patterns of VEGF165 of both origins obtained by CIEF and MALDI-MS indicate the possibility of using these analytical methods to compare samples from people with different patho- physiological conditions. This work is thus a starting point to make possible the study of the role of the various forms of VEGF165 as biomarkers. Finally, to the best of our knowledge, this is the first time that intact VEGF165 has been analyzed by CIEF and MALDI-TOF-MS.

Volatile compounds from 6 artisanal cheese varieties have been isolated by a micro-SDE (simultaneous distillation-extraction) procedure. The composition of the fractions has been anlysed by GC and GC/MS. The composition depend mainly on cheese type and ripening stage. Composition data obtained by this method seem to be useful for the characterisation of cheeses.

a-1-Acid glycoprotein (AGP) is a protein that exists in different forms, which is due to var- iations in the amino acid sequence and/or in the glycosidic part of the protein. These dif- ferences confer to these forms, among other characteristics, diverse pIs. Changes in these forms of AGP have been correlated to modifications of the pathophysiological conditions of the individuals. One of the analytical techniques employed for their study has been IEF performed in slab gels. CIEF method with hydrodynamic and chemical mobilization, in- volving an isotachophoretic process, is developed in this work to separate up to 12 bands of forms of standard AGP, which is proposed as a more reproducible, quantitative, less sam- ple-consuming, and more automated one than conventional IEF. The challenge of this work has been the development of a CIEF method for the separation of bands of a very acidic protein (pI range: 1.8–3.8) in a capillary. Intraday RSD values 1.7% have been achieved for the relative migration time of the AGP bands to that of an internal standard. For intraday area precision, RSD (%) in the range of 2.70–22.71% for AGP zones account- ing for more than 10% of total area of AGP sample has been obtained. As a proof of the potential of the methodology proposed, an AGP sample purified from a pool of sera of patients suffering from ovary cancer is analyzed by CIEF., The authors greatly acknowledge Prof. Kremmer for the kind gift of the AGP sample. This work has been supported by the Spanish CICYT, Plan I1D1I (project TIC2003-01906), by the Foundation Ramon Areces, and by the Ministry of Education and Science (Joint Action with Hungary HH04- 33). Izaskun Lacunza acknowledges the CSIC for a pre- doctoral fellowship.

High-performance frontal affinity chromatography was employed to study the adsorption–desorption kinetics characterizing the retention of Beta-lactoglobulin (Beta-LG) onto polyclonal anti-Beta-lactoglobulin (anti-Beta-LG) chromatographic supports. The adsorption and desorption processes were studied by analyzing two different elution fronts separated by a relatively long rinsing step. The method consists in performing two successive frontal injections of the protein. In between, the column was rinsed with a given volume of mobile phase (buffer alone). During this rinsing stage, a partial desorption may occur and a novel amount of protein could be adsorbed in the second frontal injection step. The whole process (first adsorption, possible desorption, and second adsorption) was simulated by a numerical procedure, in which the column was divided into a large number of slices. A model based on bi-Langmuir type kinetics was used to describe the adsorption of the protein on the support. The model assumes a non-uniform adsorbent with two types of binding sites. At equilibrium the adsorption isotherm is of the bi-Langmuir type. A global adsorption effect was considered which includes the effective binding process and the mass transfer resistances due to the transport to the binding site. Therefore, the column capacity and the kinetic parameters of the model (apparent adsorption and desorption rate constants) were determined from the best fit of the first and second adsorption fronts to the experimental ones. The other parameters of the model are the saturation capacities for the adsorption on each type of sites. The equilibrium affinity constants were estimated in a single experiment from the ratio of the apparent adsorption and desorption rate constants. The high values found (around 108 M−1) reveal a strong interaction of -LG with the immunoadsorbent. Kinetic measurements were carried out at different flow rates. Both the apparent adsorption and desorption kinetics were faster at larger flow rates, indicating an important contribution of the mass transfer resistance in the stagnant fluid at the particle boundary. However, as expected, close values were found for the resulting equilibrium constants calculated from the ratio of the apparent adsorption and desorption rate constant determined at various flow rates., A.P. acknowledges Spanish Ministry of Science and Tech- nology for a predoctoral grant. This work has been supported by Spanish CICYT (Project TIC2003-01906) and Foundation Ramon Areces. Collaboration between laboratories has been possible thanks to French-Spanish Cooperation between CNRS and CSIC.

The use of hydrolysed formulas for feeding babies is efficient for preventing allergic diseases. Several hypoallergenic formulas (HF) based on hydrolysed cows’ milk proteins have been commercialised. However, some children fed such formulas suffer allergic reactions. b-lactoglobulin (bLG) is the main allergen present in cows’ milk. In addition to the whole protein, bLG fragments have also shown allergenic character. Detection of bLG and its peptides can be a useful marker of the presence of milk in food. Enzyme linked immunoassays (ELISAs) are general methods used to analyse allergens in foods. These techniques are sensitive and selective but they have several drawbacks. Immunoassay methods performed in chromatographic systems keep the advantages of classic ELISA while avoiding the disadvantages. A sandwich enzyme linked immunoaffinity chromatography (ELIAC) method was applied to determine bLG and its peptides in several commercial HF based on different milk proteins. Samples were also fractionated by employing membranes with different molecular weight cut-offs, and the antigenicity of the peptide fractions was determined. Applying the ELIAC method, for which one analysis takes 30 min, bLG was determined at the pM level in formulas based only on caseins, and at the nM to mM level in formulas based on hydrolysed whey proteins. Peptides of molecular weight less than 3000 derived from bLG showed positive results in ELIAC assays, indicating antigenic character., A.P. acknowledges the Spanish Ministry of Science and Technology for a predoctoral grant. This work has been supported by CICYT (projects AGL2000-1480 and TIC2003-1906).

A capillary electrophoresis with laser-induced fluorescence detection method for the analysis of free amino acids (AA) in human plasma was developed. A mixture of 16 AA was on-capillary derivatized with 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ) and separated inside the capillary in less than 30 min using 70 mM borax-3.5 mM SDS pH 9.3 as running buffer. Four plasma samples from a healthy donor and patients suf- fering from phenylketonuria, propionic acidemia, and tyrosinemia type II were studied. Repeatabilities calculated as intra-day RSD (n = 3) values for the AA involved in these aminoacidopathies (glycine, phenylalanine, and tyrosine) were in the range of 0.3 to 1.2% for migration time and 3.7 to 8.2% for peak height. Reproducibilities calculated as inter-day RSD (n = 4) values for the same AA were between 0.7 and 1.4% for migration time and 4.7 and 9.1% for peak height. A fast qualitative analysis allowed the identification of the corresponding disease by comparing the electrophoretic profiles from the patient and the healthy donor and noting the increased level of the specific AA accompanying each individual disease. The results of the quantitative analysis for glycine, phenylalanine, and tyrosine in the plasma samples studied using the devel- oped method showed a good agreement with those provided by the Center of Diag- nosis of Molecular Diseases using a standard method for AA analysis., The authors acknowledge Dr. Maria Jose Garcia (Auton- oma University, Madrid, Spain) for providing plasma sam- ples and CICYT-Spain (project TIC 2003-01096) and to Ramon Areces Foundation (Spain) for financial support. M.T.V. acknowledges the Spanish Ministry of Education and Science for a pre-doctoral grant.