Abstract 2223: ELISA coupled anion exchange methodology for separation of KLK6 glycoprotein subpopulations in biological fluids
Notice bibliographique
Résumé
Abstract Ovarian cancer is the leading cause of death among all gynecological disorders. Elevated levels of Kallikrein 6 (KLK6), a secreted trypsin-like N-glycosyalted protease, have been shown to have unfavourable prognostic value in ovarian cancer patients. It has been revealed to be aberrantly up regulated at both transcriptional and translational levels in ovarian cancer. Aberrant glycosylation, or more specifically, increased sialylation of proteins has been observed in the cancer of the ovary. KLK6 isolated from ascites fluid of ovarian cancer patients has been shown to have enrichment of terminal alpha 2-6 linked sialic acid at its single N-glycosylation site when compared to protein isolated from cerebrospinal fluid (CSF), which is believed to be the major source of KLK6 present in circulation. However, due to the relatively low levels of KLK6 in serum (ng/ml range) neither mass spectrometry nor lectin based characterization of this protein found in serum has been achieved. Here, we report a novel HPLC anion exchange method, coupled to a KLK6 specific ELISA, capable of differentiating KLK6 glycoform subgroups in biological fluids, including serum, at physiologically relevant levels. In brief, biological fluids were injected onto a MonoQ bead matrix (GE Healthcare), bound proteins were eluted over a salt gradient and the resulting fractions were analyzed for KLK6 content resulting in a chromatographic elution profile containing four distinct peaks. Utilizing this assay, the KLK6 elution profile and distribution across peaks of a small set (n=8) of ovarian cancer patient matched serum and ascites fluid samples was found to be different than the profile of serum and CSF of normal individuals (n=10). Utilizing tandem mass spectrometry (MS/MS), recombinant KLK6 (rKLK6) purified from an immortalized human cell line was characterized and found to have a highly heterogeneous KLK6 population, encompassing the majority of glycoforms previously shown to be present in the protein from CSF and ovarian cancer ascites. This protein was subjected to the developed assay and was found to contain all of the four diagnostic KLK6 peaks present in the previously assayed biological fluids. Due to the available high quantity of protein from this source (mg levels) we were able to analyze the rKLK6 glycoform composition of each peak utilizing lectin affinity and MS/MS based glycopeptide quantification by single reaction monitoring. The combined results showed a significant increase in terminal alpha 2-6 linked sialic acid on the N-glycans found on KLK6 from ovarian cancer serum and ascites, as opposed to CSF and serum of normal individuals. Therefore, further development and application of this methodology might lead to improvement of KLK6 as a serum ovarian cancer biomarker. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2223. doi:10.1158/1538-7445.AM2011-2223
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Comment cette classification a été obtenuedéplier
Prédiction machine sur la base complète
Imitation des enseignantsNi prévalence calibrée, ni vérité terrain. Validation humaine à venir. Le volet Gemma est une étiquette directe du modèle pour chaque travail de la base, lue sur la notice réduite au titre. Le volet Codex est un classifieur appris des 10 348 étiquettes directes de Codex et calibré sur les taux pondérés de l'échantillon; les champs sans appui suffisant ne portent aucun appel Codex. Le mode candidate est l'union des deux volets; le consensus est leur intersection. Ces sorties portent le statut machine_predicted_unvalidated et ne sont pas des étiquettes humaines.
Scores du classifieur distillé par catégorie (deux têtes)
| Catégorie | Codex | Gemma |
|---|---|---|
| Métarecherche | 0,000 | 0,001 |
| Méta-épidémiologie (sens strict) | 0,001 | 0,000 |
| Méta-épidémiologie (sens large) | 0,000 | 0,000 |
| Bibliométrie | 0,001 | 0,000 |
| Études des sciences et des technologies | 0,000 | 0,000 |
| Communication savante | 0,001 | 0,000 |
| Science ouverte | 0,001 | 0,001 |
| Intégrité de la recherche | 0,001 | 0,001 |
| Charge utile insuffisante (le modèle a refusé de juger) | 0,003 | 0,003 |
Scores machine (provisoires)
Les deux têtes enseignantes du modèle étudiant, lues sur ce travail. Un score ordonne la base pour la relecture; il n'affirme jamais une catégorie, et le statut de validation accompagne chaque rangée tel quel.
Scores de référence d'un modèle non mature (critères de maturité non atteints, 7 itérations). Un score ordonne; il n'affirme jamais une catégorie.
score_only:v0-immature-baseline · tel quel depuis la passe de notation : score_only signifie que le nombre peut ordonner les travaux, et qu'aucune étiquette de catégorie n'en découleClassification
machine, non validéePrédiction automatique; un appel candidat d’une seule source (Gemma direct ou Codex distillé), pas un consensus.
Le détail, modèle par modèle et score par score, se trouve en fin de page sous « Comment cette classification a été obtenue ».