Trypsin Digestion Conditions of Human Plasma for Observation of Peptides and Proteins from Tandem Mass Spectrometry
Notice bibliographique
Résumé
High Resolution Image Download MS PowerPoint Slide Previous meta-analysis indicated that plasma or serum proteome groups using various experimental conditions detected different peptides from the same plasma proteins, which is strong evidence for the veracity of blood fluid LC-ESI-MS/MS but also evidences that the trypsin digestion step is a key source of variation in plasma proteomics. Agreement between different digestion conditions and MS/MS algorithms may serve as an independent confirmation of the validity of the LC-ESI-MS/MS analysis of plasma peptides. Plasma contains a high percentage of albumin held together by multiple disulfide bonds; hence, reduction and/or alkylation may greatly enhance the digestion efficiency of albumin. Plasma proteins were precipitated in 90% acetonitrile, collected over quaternary amine resin, and eluted in NaCl prior to digestion treatments. To determine the effect of trypsin digestion methods, the plasma proteins were digested in 600 mM urea and 5% acetonitrile with trypsin alone, or reduced with 2 mM DTT followed by trypsin, or DTT followed by 15 mM iodoacetamide and then trypsin. The resulting peptides were analyzed by LC-ESI-MS/MS with a linear quadrupole ion trap (LIT). The MS/MS spectra were directly fit to peptides by the X!TANDEM and SEQUEST algorithms. Blank noise injections served as the analytical control, and 30 million random MS/MS served as the statistical control. Digesting human plasma with DTT reduction, or reduction and alkylation, resulted in a dramatic increase in the number and observation frequency of albumin peptides. In contrast, digestion with trypsin alone suppressed the observation of albumin, and instead, many low abundance plasma and cellular proteins showed higher observation frequency. Digestion with trypsin alone increased the observation frequency of APOC1, ACAN, ATRN, CPB2, GP2, GPX3, HBA1, PAPD5, PKD1, and many cellular proteins. After correction against noise and random controls, SEQUEST showed good agreement with the true positive plasma proteins identified by X!TANDEM and resulted in an R -squared of 0.5238 with an F -statistic of 10,930 on 9,935 protein gene symbols with a p -value < 2.2e–16. Digestion of plasma with trypsin alone avoids the complete digestion of albumin and permits the enhanced detection of some other cellular proteins from plasma. Different digestion approaches were complimentary and together resulted in a more comprehensive plasma proteome. The protein FDR q -values, the modest effect of background and Monte Carlo correction, and the significant STRING analysis were all consistent with the high fidelity of the rigorous X!TANDEM algorithm. In contrast, SEQUEST required significant correction against noise and statistical controls and selection of high cross correlation (XCorr) scores to show good agreement with X!TANDEM. There was qualitative and quantitative agreement between plasma proteins digested without alkylation from the orbital ion trap (OIT) versus the LIT instrument that showed highly significant regression against the X!TANDEM OIT monoisotopic results, those from heavy isotopes and other masses from X!TANDEM, and with those from MaxQuant. There was significant qualitative and quantitative agreement between the complementary digestion conditions consistent with the good fidelity of plasma analysis by LC-ESI-MS/MS with a sensitive linear ion trap.
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 | 0,000 |
| Intégrité de la recherche | 0,000 | 0,000 |
| 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 ».