Mass Spectrometry
Online ISSN : 2186-5116
Print ISSN : 2187-137X
ISSN-L : 2186-5116
Technical Report
Practical Protocol for Making Calibration Curves for Direct and Sensitive Quantitative LC Orbitrap-MS of Large Neuropeptides
Tohru Yamagaki Takashi Yamazaki
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2020 Volume 9 Issue 1 Pages A0087


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.

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© 2020 Tohru Yamagaki and Takashi Yamazaki. This is an open access article distributed under the terms of Creative Commons Attribution License, which permits use, distribution, and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.
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