Practical Protocol for Making Calibration Curves for Direct and Sensitive Quantitative LC Orbitrap-MS of Large Neuropeptides.
| Autor: | Yamagaki T; Suntory Institute for Bioorganic Research, Suntory Foundation for Life Sciences., Yamazaki T; Suntory Institute for Bioorganic Research, Suntory Foundation for Life Sciences. |
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| Jazyk: | angličtina |
| Zdroj: | Mass spectrometry (Tokyo, Japan) [Mass Spectrom (Tokyo)] 2020; Vol. 9 (1), pp. A0087. Date of Electronic Publication: 2020 Aug 03. |
| DOI: | 10.5702/massspectrometry.A0087 |
| Abstrakt: | Peptides larger than 3-4 kDa, such as neuropeptide Y (NPY), orexin-B, and alpha-MSH, have practical issues that arise when conducting direct and sensitive quantitative liquid chromatography (LC) orbitrap-FT mass spectrometry (MS) due to their adsorption and low ionization efficiency, especially in standard solutions. A mixing solvent consisting of 0.5% trifluoroacetic acid (TFA) and 35-50% aq. acetonitrile was developed as the standard NPY for creating calibration curves, as well as a matrix to block the experimental tube surface to minimize adsorption. The mixture matrix effectively blocked non-specific adsorption of the standard peptides with tryptic digested bovine serum albumin (BSA) (small fragment peptides) and orexin-B (a large chain peptide). A sample containing 1 : 100 peptide:water was detected in the developed sample solution. Finally, 2 to 1,000 fmol/μL NPY could be analyzed quantitatively and reproducibly using conventional LC-MS. Parameters of the calibration curves, such as X-intercept, Bias (%), and relative standard deviation (RSD), were adjusted to optimize the sample solutions and the sensitive and quantitative LC-MS analyses. (Copyright © 2020 Tohru Yamagaki and Takashi Yamazaki.) |
| Databáze: | MEDLINE |
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