Mass Spectrometry
Online ISSN : 2186-5116
Print ISSN : 2187-137X
ISSN-L : 2186-5116

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Practical Protocol for Making Calibration Curves for Direct and Sensitive Quantitative LC Orbitrap-MS of Large Neuropeptides
Tohru YamagakiTakashi Yamazaki
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JOURNAL FREE ACCESS Advance online publication
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Article ID: A0087

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Abstract
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/L 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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