Generic approach for the sensitive absolute quantification of large undigested peptides in plasma using a particular liquid chromatography-mass spectrometry setup.
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.
Quantitative Mass Spectrometry Facility, University Hospital of Lausanne, CHUV, BH18-228, 1011 Lausanne, Switzerland.
Hamid Reza
HR
Sobhi
Bilgin
B
Vatansever
Arno
A
Wortmann
Eric
E
Grouzmann
Bertrand
B
Rochat
D000097:Acetonitriles / Q000737:chemistry; D002853:Chromatography, Liquid / Q000295:instrumentation / Q000379:methods*; D004867:Equipment Design; D006801:Humans; D013058:Mass Spectrometry / Q000295:instrumentation / Q000379:methods*; D008970:Molecular Weight; D036361:Peptide Hormones / Q000097:blood* / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D014462:Ultrafiltration
D000097:Acetonitriles; D036361:Peptide Hormones; C032159:acetonitrile