Yamagaki Tohru, Yamazaki Takashi
Suntory Institute for Bioorganic Research, Suntory Foundation for Life Sciences.
Mass Spectrom (Tokyo). 2020;9(1):A0087. doi: 10.5702/massspectrometry.A0087. Epub 2020 Aug 3.
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.
大于3-4 kDa的肽，如神经肽Y（NPY）、食欲素B和α-促黑素，在进行直接且灵敏的定量液相色谱（LC）-轨道阱傅里叶变换质谱（MS）分析时会出现实际问题，因为它们存在吸附现象且电离效率低，尤其是在标准溶液中。一种由0.5%三氟乙酸（TFA）和35-50%水相乙腈组成的混合溶剂被开发用作标准NPY来创建校准曲线，同时作为一种基质来封闭实验管表面以尽量减少吸附。该混合基质有效地阻止了标准肽与经胰蛋白酶消化的牛血清白蛋白（BSA）（小片段肽）和食欲素B（大链肽）的非特异性吸附。在开发的样品溶液中检测到了含有1:100肽:水的样品。最后，使用传统的LC-MS能够对2至1000 fmol/μL的NPY进行定量且可重复的分析。校准曲线的参数，如X轴截距、偏差（%）和相对标准偏差（RSD），经过调整以优化样品溶液以及灵敏且定量的LC-MS分析。
