In vitro quantification of follicle stimulating hormone (a Macroheterogeneity protein) in human serum using mass spectrometry is challenging and as yet unreported due to its presence in low abundance and high matrix interferences. In the present work, we identified three signature peptides for quantification of FSH using LC–MS/MS and more importantly optimized the whole serum digestion procedure enabled by Solid phase extraction (SPE) for peptide extraction. Signature peptides “AYPTPLR”, “VTVMGGFK” and “ELVYETVR”, were identified as substantial and abundant sequences for maximum sensitivity and selectivity. Tryptic digestion procedure was optimized for Human serum samples spiked with FSH in various concentrations coupled with SPE protocol to enrich signature peptide for maximal recovery. The lower limit of quantification (LLOQ) of FSH using the MRM method was 25 mIU mL⁻¹, with a linear dynamic range of 25–5000 mIU mL⁻¹ in human serum with a correlation coefficient of > 0.99. Precision and accuracy of the three batches were found to be 12.5% and 98.8% at LLOQ, 12.4% and 99.6% at low quality control (LQC), 12.5% and 97.7% at middle quality control (MQC), and 12.5% and 95.5% at high quality control (HQC), respectively. Signature peptide-based highly selective and sensitive LC–MS/MS method developed in human serum may open future applications for absolute quantification of FSH in clinical samples.

使用质谱法对人血清中的促卵泡激素（一种大异质蛋白）进行体外定量具有挑战性，并且由于其存在低丰度和高基质干扰而尚未报道。在目前的工作中，我们使用 LC-MS/MS 鉴定了三种用于 FSH 定量的特征肽，更重要的是优化了通过固相萃取 (SPE) 进行肽提取的全血清消化过程。特征肽“AYPTPLR”、“VTVMGGFK”和“ELVYETVR”被鉴定为具有最大灵敏度和选择性的大量且丰富的序列。针对掺入不同浓度 FSH 的人血清样品优化胰蛋白酶消化程序，并结合 SPE 方案以富集特征肽以获得最大回收率。^-1，在人血清中线性动态范围为 25–5000 mIU mL ^-1，相关系数 > 0.99。发现三个批次的精密度和准确度在 LLOQ 为 12.5% 和 98.8%，在低质量控制 (LQC) 为 12.4% 和 99.6%，在中等质量控制 (MQC) 为 12.5% 和 97.7%，以及 12.5% 和 95.5 % 分别在高质量控制 (HQC) 中。在人血清中开发的基于特征肽的高选择性和高灵敏度 LC-MS/MS 方法可能为临床样品中 FSH 的绝对定量开辟未来的应用。