Generic approach for the sensitive absolute quantification of large undigested peptides in plasma using a particular liquid chromatography-mass spectrometry setup.
Data(s) |
2011
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Resumo |
A generic LC-MS approach for the absolute quantification of undigested peptides in plasma at mid-picomolar levels is described. Nine human peptides namely, brain natriuretic peptide (BNP), substance P (SubP), parathyroid hormone 1-34 (PTH), C-peptide, orexines A and B (Orex-A and -B), oxytocin (Oxy), gonadoliberin-1 (gonadothropin releasing-hormone or luteinizing hormone-releasing hormone, LHRH) and α-melanotropin (α-MSH) were targeted. Plasma samples were extracted via a 2-step procedure: protein precipitation using 1vol of acetonitrile followed by ultrafiltration of supernatants on membranes with a MW cut-off of 30 kDa. By applying a specific LC-MS setup, large volumes of filtrates (e.g., 2×750 μL) were injected and the peptides were trapped on a 1mm i.d.×10 mm length C8 column using a 10× on-line dilution. Then, the peptides were back-flushed and a second on-line dilution (2×) was applied during the transfer step. The refocalized peptides were resolved on a 0.3mm i.d. C18 analytical column. Extraction recovery, matrix effect and limits of detection were evaluated. Our comprehensive protocol demonstrates a simple and efficient sample preparation procedure followed by the analysis of peptides with limits of detection in the mid-picomolar range. This generic approach can be applied for the determination of most therapeutic peptides and possibly for endogenous peptides with latest state-of-the-art instruments. |
Identificador |
https://serval.unil.ch/?id=serval:BIB_5BFBC7FA107E isbn:1873-3778 (Electronic) pmid:22018479 doi:10.1016/j.chroma.2011.09.072 isiid:000297380600009 |
Idioma(s) |
en |
Fonte |
Journal of Chromatography. A, vol. 1218, no. 47, pp. 8536-8543 |
Palavras-Chave | #Acetonitriles/chemistry; Chromatography, Liquid/instrumentation; Chromatography, Liquid/methods; Equipment Design; Humans; Mass Spectrometry/instrumentation; Mass Spectrometry/methods; Molecular Weight; Peptide Hormones/blood; Peptide Hormones/isolation & purification; Sensitivity and Specificity; Ultrafiltration |
Tipo |
info:eu-repo/semantics/article article |