No description available.

The antibody Immunoglobulin G (IgG) main function is to protect and prevent the body from infections, and it is normally found in human serum. This study is about IgG glycosylation, which is associated with different types of diseases such as neurological diseases, cancers and immunodeficiency etc. This study attempts to optimize IgG glycopeptide enrichment in a 100 μL micropipette tip set up, and to separate the enriched glycopeptides using capillary electrophoresis (CE). Matrix-assisted laser desorption/ionization – mass spectrometry (MALDI-MS) was used for data acquisition and glycopeptide profiling.  In this study, loading solutions with different combinations of acetonitrile (ACN) and trifluoroacetic acid (TFA), together with various precondition and sample preparation procedures were evaluated on IgG digest samples. Best enrichment performance, particularly regarding the selectivity, was achieved using the parameters as follows: loading solution of 83% ACN/16% H2O/1% TFA, sample solution in H2O containing 83% ACN, using a 100 μL micropipette tip packed with 1 mg cotton wool. A re-enrichment step was carried out on enriched glycopeptide samples, and improved selectivity of glycopeptides could be observed. Enriched glycopeptides could be separated into three major groups by CE using an acidic background electrolyte of 50 mM formic acid and 50 mM acetic acid, pH 2.5. / Huvudfunktionen för antikroppen Immunoglobulin G (IgG) är att skydda kroppen och förhindra infektioner och det finns normalt i mänskligt serum. Denna studie handlar om glykosylering, som är kopplad till olika typer av sjukdomar såsom neurologiska sjukdomar, cancer och immunbrist etc, och är en potentiell biomarkör för sjukdomar. Denna studie försöker optimera en IgG glycopeptidanrikningsmetod i en 100 μL mikropipettspets och separera de anrikade och intakta glycopeptiderna med hjälp av kapillärelektrofores (CE). Matrisassisterad laserdesorption/jonisering – masspektrometri (MALDI-MS) användes för datainsamling och glykopeptidprofilering  I denna studie utvärderades lösningar med olika kombinationer av acetonitril (ACN) och trifluororättiksyra (TFA) tillsammans med olika föberedelseförfaranden och provberedningsprocedurer på IgG prover. Anrikningsprestanda, särskilt selektiviteten, uppnåddes bäst med användning av följande parametrar: lösningen av 83% ACN / 16% H2O / 1% TFA, provlösning i H2O innehållande 83% ACN, med användning av en 100 mikroliter mikropipettspets fylld med 1 mg bomull. Återreningssteget genomfördes på anrikade glykopeptidprover och förbättrad selektivitet för glykopeptider kunde observeras. Anrikade glykopeptider kunde separeras i tre huvudgrupper med CE med användning av sur bakgrundselektrolyt med 50 mM myrsyra och 50 mM ättiksyra, pH 2,5.

Submitted by Haroudo Xavier Filho (haroudo.xavierfo@ufpe.br) on 2016-04-01T16:41:18Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Priscila.pdf: 3187592 bytes, checksum: a982eac78d937dd89e264fd88f2d7be9 (MD5) / Made available in DSpace on 2016-04-01T16:41:18Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Priscila.pdf: 3187592 bytes, checksum: a982eac78d937dd89e264fd88f2d7be9 (MD5) Previous issue date: 2015-02-26 / FACEPE / O câncer de próstata (CaP) é um dos tipos de tumor maligno mais incidente na população mundial, sendo também uma das principais causas de mortalidade por câncer. Geralmente ocorre em homens idosos e não é percebido em seus estágios iniciais. Para detectar o câncer de próstata, é de grande importância a avaliação pela dosagem sérica do antígeno específico da próstata (PSA), considerado o marcador tumoral da próstata. Porém, alterações nos níveis séricos do PSA são observadas também em doenças benignas da próstata, reduzindo a sensibilidade e especificidade do teste para se obter o diagnóstico. Uma peculiaridade do câncer que pode ser relevante no diagnóstico é a alteração no padrão de glicosilação de glicoproteínas secretadas e de superfície celular. A identificação de mudanças na expressão de glicanos permite a detecção precoce de vários tipos de câncer, inclusive o câncer de próstata. As lectinas são ferramentas empregadas para detectar tais alterações, graças à propriedade de reconhecer carboidratos com especificidade. Uma grande novidade no campo de estudos com lectinas é o desenvolvimento de pequenos dispositivos conhecidos como biossensores eletroquímicos baseados em lectinas para análise do perfil de glicoproteínas em processos fisiopatológicos. Esses dipositivos são elaborados utilizando eletrodos geralmente modificados com polímeros e nanomateriais, onde a lectina pode ser imobilizada e sua interação com o carboidrato quantificada com elevada sensibilidade e em tempo real. Neste trabalho foi realizado um estudo bioeletroquímico da lectina de Cratylia mollis (CramoLL) para caracterizar o padrão de glicosilação em soros de indivíduos saudáveis, com hiperplasia benigna da próstata (HBP) e CaP, utilizando biossensor eletroquímico baseado em CramoLL, imobilizada sobre eletrodo de carbono vítreo modificado com o polímero catiônico poli-Llisina (PLL) e nanotubos de carbono (NTC). Análises eletroquímicas e ópticas mostraram a formação de um filme de PLL e NTC na superfície eletródica, onde os NTC proporcionaram um aumento na área sensora de 71,2%, e nos valores de corrente anódica e catódica, e aumentaram a estabilidade da superfície, graça às propriedades físico-químicas dessas nanoestruturas. Foram investigadas as concentrações ótimas de PLL e NTC. CramoLL foi imobilizada com sucesso, numa concentração ótima de 200 μg mL-1. A interação entre CramoLL e metil-α-D-manopiranose (MeαMan)/ fetuína foi confirmada utilizando a técnica de voltametria de onda quadrada, mostrando que a lectina manteve sua atividade biológica após imobilização. Foram obtidas curvas de calibração para diferentes concentrações dos glicanos, com faixa linear de 0,96 e 38,4 μg mL-1 de MeαMan (r= 0,982, p < 0,001) e limite de detecção (LD) de 0,04 μg mL-1e de 0,5 a 25 μg mL-1 de fetuína (r= 0,994, p< 0,001) e LD = 0,05 μg mL-1. O biossensor baseado em CramoLL foi exposto à amostras de soro de indivíduos sadios (controle), com HBP e CaP com escores de Gleason 6, 7 e 9 para analisar a interação entre CramoLL e glicoproteínas dos soros de cada grupo. Os sinais de correntes obtidos por voltametria de onda quadrada mostraram diferenças significativas entre o grupo controle e os grupos HBP, CaP escore 6 e 7; entre HBP e CaP escore 6; entre CaP escore 6 e os grupos CaP escore 7 e 9 e entre CaP escore 7 e CaP 9. O potencial de reconhecimento da lectina permitiu identificar alterações de glicosilação associadas ao câncer de próstata e distinguir das amostras não cancerosas e entre os diferentes escores de CaP. Esses resultados sugerem a possibilidade de aplicação em estudos futuros envolvendo caracterização de glicosilação em câncer. / Prostate cancer (PCa) is a most frequently type of malignancy in global population and is also a major cause of cancer mortality. It usually occurs in older men and is not perceived in early stages. To detect the prostate cancer, this is of great importance the evaluation of serum prostate specific antigen (PSA), considered the prostate tumor marker. But, changes in serum PSA levels are also observed in benign prostate diseases, reducing the sensitivity and specificity of the test to obtain the diagnosis. A peculiarity of cancer that may be relevant in the diagnosis is the change in the pattern of glycosylation in glycoproteins secreted and cell surface. The identification of changes in the expression of glycans permits detection early of various types of cancer, including prostate cancer. Lectins are tools used to analyze such changes in diseases, through to the property to recognize carbohydrates with specificity. A great innovation in the field of studies with lectins is the development of smalls devices known as electrochemical biosensors based on lectins for analysis of the glycoproteins profile in physiopathological processes. These devices are typically produced using modified electrodes containing polymers, nanomaterials, where in the lectin may be immobilized and the interaction with carbohydrate quantified with high sensitivity in real time. This work presents a bioelectrochemical study of lectin of Cratylia mollis (CramoLL) to characterize the glycosylation pattern in sera from healthy individuals, with benign prostatic hyperplasia (BPH) and PCa, using electrochemical biosensor based on CramoLL immobilized on glassy carbon electrode modified with the polymer cationic poly-L-lysine (PLL) and carbon nanotubes (CNT). Electrochemical and optical analysis showed the formation of a PLL and CNT film on the electrode surface, where the CNT provided an increase in sensor area of 71.2%, and the anodic and cathodic current values, and increased stability of the surface due to the physical and chemical properties of these nanostructures. The optimal concentrations of PLL and NTC were investigated. CramoLL was successfully immobilized at a great concentration of 200 μg mL-1. The interaction between CramoLL and methyl-α-Dmannopyranose (MeαMan)/ fetuin was confirmed using voltammetry square wave technique, showing that the lectin retained its biological activity after immobilization. Calibration curves were plotted for different concentrations of the glycans with a linear range of 0.96 and 38.4 μg mL-1 for MeαMan (r = 0.982, p <0.001) and limit of detection (LOD) of 0.04 μg mL-1, and from 0.5 to 25 μg mL-1 for fetuin (r = 0.994, p <0.001) and LOD = 0.05 μg mL-1. The CramoLL-based biosensor was exposed to serum samples from healthy individuals (control), and patients with BPH and PCa with Gleason scores of 6, 7 and 9 to analyze the interaction between CramoLL and glycoproteins of sera from each group. The signals of current by square wave voltammetry show significants differents between the control group and the groups of BPH, PCa score 6 and 7; between BPH and PCa score 6; between PCa score 6 and groups PCa score 7 and 9 and between PCa score 7 ad PCa score 9. The potential of specific recognition of the lectin allowed to identify changes in glycosylation associated at prostate cancer and to distinguish of non-cancerous samples and between the differents scores of PCa. The results suggest the possibility of using in future studies involved characterization of glycosylation in cancer.

No description available.

Structure and functional study of glycans are highly challenging due to the difficulties in analyzing glycans and limited availability of samples for study. These limitations could be resolved by attaching DNA barcode to the glycan, which virtually represent glycan in further application, by increasing the sensitivity of detection by polymerase chain reaction (PCR), requiring minimal samples for analysis. Assuming bigger arena of DNA Encoded Glycan Libraries (DEGL) in future, we propose here a method for uniquely coding all glycans using computer program that can convert the structural information of glycans to DNA barcode. A unique and universal coding for glycans will benefit both synthesis and analysis of DEGLs. As a proof of principle study, a small DNA Encoded Glycan Library (DEGL) of blood and globo series glycan antigen and its application was demonstrated in detecting blood group and breast cancer from plasma.

Oligosaccharides play very crucial biological roles in the human body. They are structurally diverse and very challenging to analyze. In an attempt to contribute towards the analysis of oligosaccharides in the growing filed of 'Glycomics', we explored different ways to make novel efficient oligosaccharide tags to label N -linked oligosaccharides from glycoproteins. We synthesized and characterized various oligosaccharide tags which are useful to analyze the oligosaccharide by mass spectrometry, HPLC, and bioaffinity. In these tags we incorporated ar UV/fluorescent core, a biotin moiety and an amino/azido/alkyne terminus. We varied the structures of the tags for improved solubility in common organic solvents. These tags were used to label standard oligosaccharides, and the labeling efficiency was evaluated.

La glycosylation des protéines est une modification co/post-traductionnelle, localiséeprincipalement dans l’appareil de golgi, impliquée dans divers processus physiologiques. Àl’inverse de la synthèse des protéines et des nucléotides, celle des sucres est plus complexe,en partie, à cause des nombreux branchements structuraux et de la diversité stéréochimiquedes glycannes. Les glycosyltransférases sont les enzymes responsables de la biosynthèse desoligo- et polysaccharides. Cependant, il existe différents procédés permettant de réaliser lasynthèse in vitro d’oligosaccharides tels que des procédés chimiques, enzymatiques ouchimio-enzymatiques. La Core 2 β(1,6)-N-acétylglucosaminyltransférase I (C2GnT-I) est uneglycosyltransférase (GT) transmembranaire de type II qui crée une liaison β1,6 entre une Nacétylglucosamine(GlcNAc) et la N-acétylgalactosamine (GalNAc) d’un noyau Core 1,formant ainsi une structure branchée de type Core 2. Une fois le branchement Core 2 initié,au moins trois réactions successives de transfert peuvent avoir lieu, impliquant les β(1,4)-galactosyltransférases, les α(2,3)-sialyltransférases et les α(1,3)-fucosyltransférases. Il estadmis que les GTs sont réparties séquentiellement dans les compartiments cis, médian ettrans de l’appareil de Golgi selon leur ordre d’intervention et le type cellulaire. Nous avonsmis au point un procédé de reconstitution membranaire, comportant des glycoprotéinesgolgiennes, dont les GTs, par l’intermédiaire d’une lectine, la wheat germ agglutinin (WGA).Il est admis que la WGA interagit avec les composants de la membrane plasmique et ceux del’appareil de Golgi. L’étude de notre système membranaire reconstitué a mis en évidence uneactivité élevée de la C2GnT-1 in vitro, et son efficacité à synthétiser des oligosaccharidesbranchés Core 2, par glycosylation séquentielle. / Protein glycosylation is a co/posttranslational modification, localized in the Golgi apparatus,involved in various physiological processes. Sugar synthesis is more complex than that ofproteins and nucleic acids, in part because of the glycosidic bond and glycan stereochemistrydiversity. Glycosyltransferases are enzymes responsible of oligo- and polysaccharidesbiosynthesis. Various methods are used for the synthesis of oligosaccharides, in vitro, suchas chemical, enzymatic or chemo-enzymatic. Core 2 β(1,6)-N-acetylglucosaminyltransferase I(C2GnT-I) is a type-II transmembrane glycosyltransferase (GT). This enzyme create a β1,6bond between N-acetylglucosamine (GlcNAc) and Core 1 N-acetylgalactosamine (GalNAc),forming a Core 2 branched structure. Once the Core 2 branch is initiated, at least threesuccessive transfer reactions can take place, involving β(1,4)-galactosyltransferases, α(2,3)-sialyltransferases and α(1,3)-fucosyltransferases. It is known that GT are distributedsequentially in the cis, medial and trans Golgi apparatus in order of intervention andaccording to cell type. We have developed a membrane reconstitution process comprisinggolgi glycoproteins, including GT, by the use of a lectin, the wheat germ agglutinin (WGA). Itis known that WGA interacts with plasma membrane and Golgi apparatus components. Ourreconstituted membrane system showed a high C2GnT-1 activity in vitro and its effectivenessin synthesizing Core2 branched oligosaccharides by sequential glycosylation.

Glykane aus exokrinen Drüsen stellen ein Schutzsystem der Schleimhautoberflächen dar, indem sie an mikrobielle Adhäsine binden und dadurch Einfluß auf die mikrobielle Besiedelung und Invasion des Wirtes nehmen. 11 Patienten mit rasch fortschreitender Parodontitis (RPP) wurde über jeweils 20 Minuten in Ruhe und unter adrenerger Belastung Speichel aus den großen Speicheldrüsen entnommen und in einem kompetitiven Lektinbindungsinhibitionstest auf die Bindungsfähigkeit an 8 verschiedene Planzenlektine untersucht. Patienten mit RPP zeigen ein anderes Verteilungsmuster der antiadhäsiven Glycane als die Kontrollgruppe. Sie zernieren u.a. aus den Unterkieferdrüsen konstant signifikant mehr Glykane mit endständigen Mannosegruppen, aus der Parotis dagegen nur unter adrenerger Stimulation. Dies zeigt die unterschiedliche Funktion der Drüsen bei der Beeinflussung des Milieus in der Mundhöhle als auch einen unterschiedlichen Erregungszustand des Schleimhautschutzsystems bei Erkrankten und Kontrollen. Aus den Ergebnissen können sich diagnostische Aspekte für das Risiko und den Aktivitätszustand einer parodontalen Erkrankung ergeben. / Glycans from exocrine glands create a defense system of the mucosal surfaces by binding to microbial adhesins and interfering with colonisation and invasion of the host. Saliva from 11 patients with rapidly progressive periodontitis (RPP) was collected over a period of 20 minutes each in rest and during adrenergic stimulation. The samples were tested for binding properties to 8 different plant lectins by a competitive lectin binding inhibition test. A pattern of antiadhesive glycans different from the control group is secreted in patients with RPP. The latter constantly secrete significantly more glycans with terminal mannose from the mandibular glands. In the parotis this is only the case during adrenergic stimulation. This demonstrates the different purpose of the glands in maintaining the oral milieu as well as different states of activity of the mucosal defense system in RPP patients and controls. Diagnostic aspects for risk and activity of periodontal diseases can be drawn from these findings.

A prolactina humana (hPRL) é um hormônio polipeptídico secretado pela hipófise anterior sob regulação do hipotálamo, envolvido em uma variedade de processos biológicos como o desenvolvimento da glândula mamária e lactação. O produto recombinante é importante no diagnóstico médico e no tratamento de insuficiência da lactação. Este hormônio pode ocorrer sob a forma de proteína não glicosilada (NG-hPRL) e glicosilada (G-hPRL), com pesos moleculares de aproximadamente 23 e 25 kilodalton (kDa), respectivamente; possui um único sítio de N-glicosilação localizado na asparagina (Asn) posição 31, que é parcialmente ocupado, representando assim um modelo particularmente interessante de glicosilação. A atividade biológica da G-hPRL é muito menor comparada à NG-hPRL (~4 vezes) e sua função fisiológica ainda não é bem definida: a porção de carboidrato parece ter um importante papel na biossíntese, secreção, atividade biológica, e sobrevivência plasmática do hormônio. O objetivo principal desse trabalho foi comparar as estruturas dos N-glicanos presentes na prolactina glicosilada hipofisária (G-hPRL-NHPP) com a recombinante. Para obter a G-hPRL recombinante foi realizada uma produção em escala laboratorial a partir de células de ovário de hamster chinês (CHO) geneticamente modificadas e adaptadas ao crescimento em suspensão. Foi adicionada, ao meio de cultura cicloheximida (CHX), cujo efeito principal foi aumentar a relação G-hPRL/NGhPRL que passou de 5% para 38%, facilitando assim a purificação da G-hPRL. A G-hPRL foi purificada em duas etapas, uma troca catiônica seguida de purificação por cromatografia liquida de alta eficiência de fase reversa (RP-HPLC) que se demonstrou eficiente na separação das duas isoformas de hPRL. A G-hPRL recombinante IPEN foi assim analisada por diversas técnicas confirmando a sua pureza e atividade biológica, incluindo comparações com outras amostras de referências de origem hipofisária adquirida junto ao National Hormone & Peptide Program (NHPP-E.U.A.) . Foi realizada também a determinação inédita de Nglicanos presentes na G-hPRL produzida por células CHO e na G-hPRL nativa, produzida pela hipófise humana, possibilitando comparar as duas estruturas de carboidratos e alcançando assim uma das principais metas desse projeto. Entre as principais diferenças encontradas nas estruturas dos dois N-glicanos, destacam-se a baixa quantidade de ácido siálico (NeuAc), a alta porcentagem de glicanos sulfatos (74,0%) e com fucose (Fuc) (93,3%) presentes na amostra hipofisária e a tendência da preparação recombinante de apresentar glicanos com maior peso molecular e com uma menor variação nas isoformas. / Human prolactin (hPRL) is a polypeptide hormone secreted by the anterior pituitary under the regulation of the hypothalamus, involved in a variety of biological processes such as mammary gland development and lactation. The recombinant product is important in medical diagnosis and treatment of failure of lactation. This hormone may occur in the form of non-glycosylated protein (NGhPRL) and glycosylated (G-hPRL) with molecular weights of approximately 23 and 25 kilodalton (kDa), respectively; has a single N-glycosylation site located at asparagine (Asn) position 31, which is partially occupied, thus being a particularly interesting model of glycosylation. The biological activity of G-hPRL is lower compared to NG-hPRL (~4 times) and its physiological function is not well defined: the portion of carbohydrate appears to have an important role in the hormone biosynthesis, secretion, biological activity, and plasma survival of the hormone. The main objective of this study was to compare the structures of N-glycans present in glycosylated pituitary prolactin (G-hPRL-NHPP) with those present in the recombinant. To obtain the recombinant G-hPRL the production was performed in laboratory scale from Chinese hamster ovary cells (CHO), genetically modified and adapted to growth in suspension. Cycloheximide (CHX), whose main effect was to increase the ratio G-hPRL/NG-hPRL from 5% to 38% was added to the culture medium, thereby facilitating the purification of G-hPRL. The G-hPRL was purified in two steps, a cation exchanger followed by a purification by reversed-phase high performance liquid chromatography (RP-HPLC) which demonstrated the efficient separation of the two isoforms of hPRL. Recombinant G-hPRL-IPEN was well characterized by several techniques confirming its purity and biological activity, including comparisons with other reference preparation of pituitary origin purchased from the \"National Hormone & Peptide Program (NHPPU. S.)\". The composition of N-glycans present in the G-hPRL, produced by CHO cells, and that of native G-hPRL, produced by the human pituitary gland, were also determined for the first time, allowing the two structures of carbohydrates to be compared and thus, achieving one of the main goals of this project. Among the main differences in N-glycan structures, we highlight the low presence of sialic acid (NeuAc) and the high percentage of sulfated glycans (74.0%) and of fucose (Fuc) (93.3%) in the pituitary sample and the tendency of the recombinant preparation to present glycans with higher molecular weight and less isoforms variation.

A prolactina humana (hPRL) é um hormônio polipeptídico secretado pela hipófise anterior sob regulação do hipotálamo, envolvido em uma variedade de processos biológicos como o desenvolvimento da glândula mamária e lactação. O produto recombinante é importante no diagnóstico médico e no tratamento de insuficiência da lactação. Este hormônio pode ocorrer sob a forma de proteína não glicosilada (NG-hPRL) e glicosilada (G-hPRL), com pesos moleculares de aproximadamente 23 e 25 kilodalton (kDa), respectivamente; possui um único sítio de N-glicosilação localizado na asparagina (Asn) posição 31, que é parcialmente ocupado, representando assim um modelo particularmente interessante de glicosilação. A atividade biológica da G-hPRL é muito menor comparada à NG-hPRL (~4 vezes) e sua função fisiológica ainda não é bem definida: a porção de carboidrato parece ter um importante papel na biossíntese, secreção, atividade biológica, e sobrevivência plasmática do hormônio. O objetivo principal desse trabalho foi comparar as estruturas dos N-glicanos presentes na prolactina glicosilada hipofisária (G-hPRL-NHPP) com a recombinante. Para obter a G-hPRL recombinante foi realizada uma produção em escala laboratorial a partir de células de ovário de hamster chinês (CHO) geneticamente modificadas e adaptadas ao crescimento em suspensão. Foi adicionada, ao meio de cultura cicloheximida (CHX), cujo efeito principal foi aumentar a relação G-hPRL/NGhPRL que passou de 5% para 38%, facilitando assim a purificação da G-hPRL. A G-hPRL foi purificada em duas etapas, uma troca catiônica seguida de purificação por cromatografia liquida de alta eficiência de fase reversa (RP-HPLC) que se demonstrou eficiente na separação das duas isoformas de hPRL. A G-hPRL recombinante IPEN foi assim analisada por diversas técnicas confirmando a sua pureza e atividade biológica, incluindo comparações com outras amostras de referências de origem hipofisária adquirida junto ao National Hormone & Peptide Program (NHPP-E.U.A.) . Foi realizada também a determinação inédita de Nglicanos presentes na G-hPRL produzida por células CHO e na G-hPRL nativa, produzida pela hipófise humana, possibilitando comparar as duas estruturas de carboidratos e alcançando assim uma das principais metas desse projeto. Entre as principais diferenças encontradas nas estruturas dos dois N-glicanos, destacam-se a baixa quantidade de ácido siálico (NeuAc), a alta porcentagem de glicanos sulfatos (74,0%) e com fucose (Fuc) (93,3%) presentes na amostra hipofisária e a tendência da preparação recombinante de apresentar glicanos com maior peso molecular e com uma menor variação nas isoformas. / Human prolactin (hPRL) is a polypeptide hormone secreted by the anterior pituitary under the regulation of the hypothalamus, involved in a variety of biological processes such as mammary gland development and lactation. The recombinant product is important in medical diagnosis and treatment of failure of lactation. This hormone may occur in the form of non-glycosylated protein (NGhPRL) and glycosylated (G-hPRL) with molecular weights of approximately 23 and 25 kilodalton (kDa), respectively; has a single N-glycosylation site located at asparagine (Asn) position 31, which is partially occupied, thus being a particularly interesting model of glycosylation. The biological activity of G-hPRL is lower compared to NG-hPRL (~4 times) and its physiological function is not well defined: the portion of carbohydrate appears to have an important role in the hormone biosynthesis, secretion, biological activity, and plasma survival of the hormone. The main objective of this study was to compare the structures of N-glycans present in glycosylated pituitary prolactin (G-hPRL-NHPP) with those present in the recombinant. To obtain the recombinant G-hPRL the production was performed in laboratory scale from Chinese hamster ovary cells (CHO), genetically modified and adapted to growth in suspension. Cycloheximide (CHX), whose main effect was to increase the ratio G-hPRL/NG-hPRL from 5% to 38% was added to the culture medium, thereby facilitating the purification of G-hPRL. The G-hPRL was purified in two steps, a cation exchanger followed by a purification by reversed-phase high performance liquid chromatography (RP-HPLC) which demonstrated the efficient separation of the two isoforms of hPRL. Recombinant G-hPRL-IPEN was well characterized by several techniques confirming its purity and biological activity, including comparisons with other reference preparation of pituitary origin purchased from the \"National Hormone & Peptide Program (NHPPU. S.)\". The composition of N-glycans present in the G-hPRL, produced by CHO cells, and that of native G-hPRL, produced by the human pituitary gland, were also determined for the first time, allowing the two structures of carbohydrates to be compared and thus, achieving one of the main goals of this project. Among the main differences in N-glycan structures, we highlight the low presence of sialic acid (NeuAc) and the high percentage of sulfated glycans (74.0%) and of fucose (Fuc) (93.3%) in the pituitary sample and the tendency of the recombinant preparation to present glycans with higher molecular weight and less isoforms variation.