Proline/Alanine Tails as Ionization Enhancement Tags in Native Mass Spectrometry
Notice bibliographique
Résumé
Introduction: Asparaginase (ASNase) is an enzyme used for the treatment of acute lymphoblastic leukemia (ALL) and is an integral component of treatment protocols with long-term survival rates in children that have progressively increased to about 90%. We are presently carrying out native mass spectrometry, ion mobility and hydrogen deuterium exchange on a ‘PASylated’ version of this protein, which incorporates a Proline/Alanine ‘tail’ (Biopolymers 2018, 109, e23069) in order to enhance longevity in vivo using PASylation technology. While characterizing structural features of this protein that confer resistance to degradation in vivo, we noted that this species exhibits a unique electrospray ionization mechanism that appears to be driven primarily by chain ejection, even for the relatively large, folded tetramer. In this work, we explore the role of a long-disordered tail in protein ionization, including the possibility that PA(S)tails may serve as ‘ionization enhancement tags’ for proteins that are inefficiently ionized via electrospray. Method: PASylated ASNase was acquired from Jazz pharmaceuticals. These batches were treated using 40 KDa MWCO Zeba desalting columns and subjected to native mass spectrometry on a Waters G2S ion mobility mass spectrometer by direct infusion ESI. Data analysis was carried out using the Unidec deconvolution software package (Anal. Chem. 2015, 87, 8, 4370–4376). Preliminary data: To better understand the structure of PA-tagged protein, we ran electrospray ionization (ESI) mass spectrometry for both the core enzyme Erwinia Asparaginase and the PASylated Asparaginase. The untagged protein exhibits a typical ‘native’ mass spectrum for a large multimeric protein, corresponding to relatively low charge and a relatively narrow distribution associated with charge residue ionization. The population is more than 90% tetrameric, based on the relative peak intensities of the tetramer vs. the monomer. The peaks associated with the monomer also appear to ionize via a charge residue mechanism, which is consistent with a folded protein. Interestingly, the PA tail, added as a PASylation tag to improve in vivo stability of this protein, radically changes the native ESI mass spectrum. In particular, the tagged protein exhibits a mixture of intense, highly charged monomeric, dimeric, trimeric and tetrameric species. This suggests that the PASylated protein has undergone ionization primarily by chain ejection rather than charge residue, which would imply that the ionization mechanism is dominated by the unstructured PA ‘tail’, in line with previous observations made with isolated PAS sequences (J. Am. Soc. Mass Spectrom. 2014 25, 1489-1497). This raises two questions that we will explore: (1) What is the minimum length of unstructured ‘tail’ required for a ‘native’ protein to favor chain ejection over charge residue and (2) can PA tails be used as ‘ionization enhancement tags’ to facilitate detection of large or otherwise ‘difficult to ionize’ protein complexes? Novel aspects: In this work, we propose PASylation ® as a new tool for ionization enhancement through chain ejection.
Récupéré en direct depuis OpenAlex et désinversé. Les résumés ne sont pas conservés dans cette base de données : les index inversés représentent 8,6 Go des 9,3 Go de texte de la base, et le serveur dispose de 13 Go libres.
Comment cette classification a été obtenuedéplier
Prédiction distillée sur la base complète
Imitation des enseignantsNi prévalence calibrée, ni vérité terrain. Validation humaine à venir. Apprise à partir de 10 348 étiquettes directes de Codex et de 10 348 étiquettes directes de Gemma. Le mode candidate est l'union des têtes enseignantes seuillées; le consensus est leur intersection. Ces sorties portent le statut machine_predicted_unvalidated et ne sont ni des étiquettes humaines ni des étiquettes directes de modèles de pointe.
Scores Codex et Gemma par catégorie
| Catégorie | Codex | Gemma |
|---|---|---|
| Métarecherche | 0,000 | 0,000 |
| Méta-épidémiologie (sens strict) | 0,000 | 0,000 |
| Méta-épidémiologie (sens large) | 0,000 | 0,000 |
| Bibliométrie | 0,000 | 0,000 |
| Études des sciences et des technologies | 0,000 | 0,000 |
| Communication savante | 0,000 | 0,000 |
| Science ouverte | 0,000 | 0,001 |
| Intégrité de la recherche | 0,001 | 0,001 |
| Charge utile insuffisante (le modèle a refusé de juger) | 0,000 | 0,000 |
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 tête enseignante, 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 ».