Oocyte development in Xenopus laevis spans six morphologically distinct stages (stage I-VI), and is associated with a decrease in protein O-GlcNAc levels. As a first step in elucidating the role of O-GlcNAc in developing oocytes, initial efforts were focused on isolation and identification of fifteen modified proteins that decrease during oocyte development. Stage I oocytes due to their high amounts of these proteins, were used as starting material for purification. Multiple affinity and specific antibody based purification technique were initially used in an attempt to enrich the O-GlcNAc proteins. Due to the unique properties of the proteins ultimately identified, these techniques were unable to provide sufficient material for sequencing. However, differential centrifugation coupled with 2D-gel electrophoresis was highly successful. The majority of isolated proteins were strongly basic in nature with pIs 8-10. Coomassie stained bands from 2D-analysis were trypsin digested, and peptides were sequenced by mass spectroscopy (Finnigan LCQ). Mass data were interpreted by Bioworks software, and protein sequences were compared to multiple protein databases. Initially, six proteins were identified as Thesaurin a (42Sp50), cytoplasmic mRNA binding protein p54, y-box homolog, Xp 54 (ATP dependent RNA helicase p54), Vg1 RNA binding protein variant A, Zygote arrest 1(Zar1) and Poly (A) binding protein (PABP). Thesaurin a, the main component of 42S particle of previtellogenic oocytes (stages I-III) is involved in tRNA storage and possess low tRNA transfer activity; y-box factor homolog and Xp54 are present in oocyte mRNA storage ribonucleoprotein particles; Vg1 RBP variant A associates mVg1 RNA to microtubules in order to translocate to the vegetal cortex; Zar1 is involved in oocyte-to-embryo transition; and PABP initiates mRNA translation. This study is the first to characterize these oocyte specific proteins as O-GlcNAc modified proteins. Overall, the presence of several O-GlcNAc proteins in oocytes, the reduction in their levels/ O-GlcNAc levels, and the variation in maturation time in the presence of HBP-flux modulators in developing oocyte indicates O-GlcNAc may play important roles in metabolism, cell growth and cell division of X. laevis oocytes. Therefore, identifying the remainder of these proteins and elucidating the O-GlcNAc role in their function is a worthwhile pursuit.

L’approche analytique d’interrogation sur puce par spectrométrie de masse est une techniqueparticulièrement bien adaptée à l’analyse multiplexée requise pour la recherche de biomarqueurs dansle diagnostic moderne. L’objectif a été de contribuer aux développements technologiques etméthodologiques d’une plateforme d’analyse baptisée SUPRA-MS (Imagerie par Résonance desPlasmons de Surface en array combinée à la Spectrométrie de Masse). Des puces d’or compatiblesavec la SPRi et la MS ont été conçues et réalisées à l’aide des techniques de dépôt sous vide. Uneétude originale couplant la SPRi avec l’AFM a permis d’établir une relation entre le signal SPRmesuré et la quantité réelle de protéines fixées sur des puces nanostructurées. Nous avons ensuitedéveloppé une procédure d’immobilisation en format array (16 à λ6 spots) par liaison covalente enmonocouche des anticorps spécifiques, dirigés contre un biomarqueur du cancer du sein (LAG3) pourune analyse multiplexée d’échantillons biologiques. Du plasma humain contenant 300 ng/mL deLAGγ est injecté à la surface de la puce. L’injection est suivie en temps réel par SPRi et conduit à unecapture du biomarqueur à l’échelle de la femtomole par spots. Un traitement collectif des spots parspray pour la digestion in situ des protéines et le dépôt de matrice en vue d’une interrogation MS enMALDI-TOF a été mis au point par l’équipe du Dr Patrick Ducoroy (CLIPP-CHU Dijon). Lesrésultats MS obtenues ont permis 100 % d’identification du biomarqueur. Cette technologie sansmarquages spécifiques est particulièrement bien adaptée à la caractérisation fine des biomarqueurs et àla discrimination de variants protéiques. / The analytic approach of interrogation on chip by mass spectrometry is a suitable technique tomultiplexed analysis required for biomarker research in modern diagnosis. The aim was to contributeto the technological and methodological developments of analysis platform called SUPRA-MS(Surface Plasmon Resonance in Array coupled to Mass Spectrometry) whose goal is to provideadditional data to assay on the target protein by mass spectrometry. At first, gold chips compatiblewith SPRi and MS were designed and fabricated using vacuum deposition techniques. An originalstudy coupling SPRi with AFM has established a relationship between the SPR signal measured andthe real amount of proteins bound to nanostructured chips. We developed an immobilization procedurein array format (spots 16-96) by covalent monolayer of specific antibodies directed against the proteinLAG3, a biomarker of breast cancer for multiplex analysis of human biological samples (plasma).Human plasma containing 300 ng/mL of LAG3 is injected to the chip surface. The injection ismonitored in real time by SPRi and leads to the biomarker capture at the femtomole scale. After thebiosensing step, a collective treatment of spots by spray for in situ protein digestion and matrixdeposition in view of a MS analysis was developed by Dr. Patrick Ducoroy's team (CLIPP-CHUDijon). The MS and MS-MS analysis by MALDI-TOF was developed to analyze all spotsautomatically and determine their peptide mapping leading to 100% of the biomarker identification.This technology does not require the use of specific markers, is suitable to the biomarkerscharacterization and discrimination of protein variants.