Robust and sensitive peptidomics workflow for plasma based on specific extraction, lipid removal, capillary LC setup and multinozzle ESI emitter.

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State: Public
Version: author
License: CC BY 4.0
Serval ID
serval:BIB_B6EB05E6B898
Type
Article: article from journal or magazin.
Collection
Publications
Institution
Title
Robust and sensitive peptidomics workflow for plasma based on specific extraction, lipid removal, capillary LC setup and multinozzle ESI emitter.
Journal
Talanta
Author(s)
Rochat B., Waridel P., Barblan J., Sottas P.E., Quadroni M.
ISSN
1873-3573 (Electronic)
ISSN-L
0039-9140
Publication state
Published
Issued date
01/02/2021
Peer-reviewed
Oui
Volume
223
Number
Pt 1
Pages
121617
Language
english
Notes
Publication types: Journal Article
Publication Status: ppublish
Abstract
We present a new workflow for the LC-MS determination of native peptides in plasma at picomolar levels. Collected whole blood was quickly diluted with an ice-cold solution in order to stop protease activity. Diluted plasma samples were extracted by protein denaturation followed by solid-phase-extraction with a polymeric stationary phase that removed most proteins and lipids. Using a specific LC-MS setup with 3 pumps, 240 μL of extracts were injected without drying-reconstitution, a step known to cause peptide losses. After an 18-fold dilution on-line, peptides were trapped on a 1 × 10 mm C8 column, back-flushed and resolved on a 0.3 × 100 mm C18 column. Extract reproducibility, robustness (column clogging), extraction yields, matrix effects, calibration curves and limits of detection were evaluated with plasma extracts and spiked-in standards. The sensitivity and applicability of 3 electrospray sources were evaluated at capillary flow rates (10 μL/min). We show that ionization sources must have a spray angle with the MS orifice when "real" extracts are injected and that a multinozzle emitter can improve very significantly peptide detection. Finally, using our workflow, we have performed a peptidomics study on dried-blood-spots collected over 65 h in a healthy volunteer and discovered 5 fragments (2.9-3.8 KDa) of the protein statherin showing circadian oscillations. This is the first time that statherin is observed in blood where its role clearly deserves further investigations. Our peptidomic protocol shows low picomolar limits of detection and can be readily applied with or without minor modifications for most peptide determinations in various biomatrices.
Keywords
Blood, High-resolution mass spectrometry, Multi-nozzle electrospray, Peptide, Plasma, Statherin
Pubmed
Web of science
Open Access
Yes
Create date
22/12/2020 11:01
Last modification date
06/01/2021 6:25
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