CIEF with hydrodynamic and chemical mobilization for the separation of forms of Æ-1-acid glycoprotein

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.Peer reviewe

CIEF with hydrodynamic and chemical mobilization for the separation of forms of α-1-acid glycoprotein

α-1-Acid glycoprotein (AGP) is a protein that exists in different forms, which is due to variations in the amino acid sequence and/or in the glycosidic part of the protein. These differences 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, involving 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 sample-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 accounting 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. © 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.Peer Reviewe

On-capillary fluorescent labeling and capillary electrophoresis laser-induced fluorescence analysis of glycoforms of intact prostate-specific antigen

Peer Reviewe

Retention parameters in chromatography (IUPAC recommendations 2001)

Contiene : Part A.Hold-up volume concept in column chromatography Part B.Retention parameters in gas chromatographyThe paper presents a revision of terms in the IUPAC "Nomenclature for Chromatography", Pure and Applied Chemistry, 65, 819-872, 1993. The terms revised pertain to hold-up volumes in gas, liquid, and supercritical-fluid chromatography, as well as to basic retention parameters, especially in gas chromatography. A number of related and derived definitions are described, including definitions of the terms "chromatographic process" and "chromatographic phase system". A number of the original terms were found to be misleading or superfluous, including such terms as corrected retention time, net retention time, total retention volume (time), and specific retention volume at 0 °C, and their use is strongly discouragedPeer reviewe

CZE of human alpha-1-acid glycoprotein for qualitative and quantitative comparison of samples from different pathological conditions

Alpha-1-acid glycoprotein (AGP) presents different forms, which may arise from diffferences in the amino acid sequence and/or in the glycosidic part of the protein. Changes in forms of AGP have been described in literature as a possible tumor marker. While most previous works have approached the study of glycopeptides and/or glycans obtained after fragmentation of the protein, in this work, a CZE method is developed to separate up to eleven peaks of intact forms of AGP. A computer program developed in our laboratory is used to select the migration parameters that make possible an accurate assignment of AGP peaks. Electropherograms of AGP samples purified from sera of cancer patients and healthy donors are qualitatively and quantitatively compared. Percentages of correct assignment of AGP peaks close to 100% are achieved by using either the migration time of each peak relative to that of the EOF marker or the effective electrophoretic mobility of the peaks. The computer program permits to select, among different hypotheses for peak allotment, that one providing the highest accuracy of assignment. In this way, some peaks with different charge-to-mass ratio and a different distribution of area percentage of AGP forms are observed when comparing samples from sick and healthy individuals. Thus, a method that permits to compare AGP forms existing in sera of individuals with different pathophysiological situations has been developed. A potential for using AGP forms analyzed by CZE as a disease marker and for using this technique for screening purposes is envisaged. © 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.Peer Reviewe

Immunoaffinity chromatographic isolation of prostate-specific antigen from seminal plasma for capillary electrophoresis analysis of its isoforms

Prostate-specific antigen (PSA) concentration in serum has been the biomarker employed for prostate cancer diagnosis in the last two decades. However, new more specific biomarkers allowing a better differentiation of cancer from non-malignant prostate diseases are necessary. Glycosylation of PSA gives rise to different forms of the protein which can be separated into several isoforms by analytical techniques, such as CE. Because PSA glycosylation is influenced by pathological conditions, the CE pattern of PSA isoforms could be different in prostate cancer than in non-malignant prostate diseases. To study this CE pattern of PSA, prior purification of the protein from the biological fluid is mandatory. In this study an immunoaffinity chromatography method which allows PSA purification without altering the CE pattern is developed. An in-house prepared column produced with commercial anti-PSA antibodies is employed. The use of 1. M propionic acid as elution agent provides higher than 40% recovery of high purity PSA. CE analysis of PSA immunopurified from seminal plasma of a healthy individual shows the same 8 peaks as the commercially available PSA standard. Sample preparation only requires dilution with phosphate buffered saline prior to immunoaffinity purification. High repeatability for the sample preparation step was achieved (RSD% for percentage of corrected peak area in the range 0.6-5.3 for CE analysis of three independently purified seminal plasma aliquots compared to range 0.8-4.9 for a given aliquot analyzed three times by CE). IAC of five microliters seminal plasma provided enough PSA to achieve signal/noise ratio larger than 5 for the smallest CE isoforms. © 2014 Elsevier B.V.Peer Reviewe

Capillary electrophoresis of glycoproteins

Authors thank the Spanish Ministry of Science and Innovation and Spanish State Research Agency (Project PID2019-106405GB-I00/AEI/10.13039/501100011033) and the Comunidad of Madrid and European funding from FSE and FEDER programs (project reference S2018/ BAA-4393, AVANSECAL-II-CM) for financial support. Laura Gomez-Ruiz acknowledges the Comunidad de Madrid for the contract in the frame of the YGI Plans financed by the ESF and the YEI

Monitorization of alfa1-Acid Glycoprotein Deglycosylation using SU-8 Microchips Electrophoresis with LIF Detection

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 ¿1-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 authors acknowledge the Spanish Ministries of Science and Innovation and of Economy, Industry, and Competitiveness (grants PSS-010000-2008-6 and CTQ2013-43236-R, respectively) and CSIC (joint project 2009JP003 with the Japanese Society for Promotion of Science) for financial support. M.M.B-R thanks the CSIC for a JAE-Pre contract and A.G.C. acknowledges CSIC for JAE-Doc contract. These contracts are co-financed by the European Union under the European Social Fund (ESF)