Proteomic Analysis of Human Plasma during Intermittent Fasting

Intermittent fasting
(IF) increases lifespan and decreases metabolic
disease phenotypes and cancer risk in model organisms, but the health
benefits of IF in humans are less clear. Human plasma derived from
clinical trials is one of the most difficult sample sets to analyze
using mass spectrometry-based proteomics due to the extensive sample
preparation required and the need to process many samples to achieve
statistical significance. Here, we describe an optimized and accessible
device (Spin96) to accommodate up to 96 StageTips, a widely used sample
preparation medium enabling efficient and consistent processing of
samples prior to LC–MS/MS. We have applied this device to the
analysis of human plasma from a clinical trial of IF. In this longitudinal
study employing 8-weeks IF, we identified significant abundance differences
induced by the IF intervention, including increased apolipoprotein
A4 (APOA4) and decreased apolipoprotein C2 (APOC2) and C3 (APOC3).
These changes correlated with a significant decrease in plasma triglycerides
after the IF intervention. Given that these proteins have a role in
regulating apolipoprotein particle metabolism, we propose that IF
had a positive effect on lipid metabolism through modulation of HDL
particle size and function. In addition, we applied a novel human
protein variant database to detect common protein variants across
the participants. We show that consistent detection of clinically
relevant peptides derived from both alleles of many proteins is possible,
including some that are associated with human metabolic phenotypes.
Together, these findings illustrate the power of accessible workflows
for proteomics analysis of clinical samples to yield significant biological
insight.

间歇性禁食（Intermittent Fasting, IF）在模式生物中可延长寿命、减轻代谢疾病表型并降低癌症风险，但间歇性禁食对人类的健康益处尚不明晰。人类临床试验来源的血浆是基于质谱的蛋白质组学（mass spectrometry-based proteomics）分析中最难处理的样本集之一，这是因为其需要复杂的样本前处理流程，且需要处理大量样本以获得统计学显著性。本研究介绍了一款经过优化且易于使用的装置（Spin96），可兼容最多96个StageTips——这是一种广泛使用的样本前处理介质，能够在液相色谱-串联质谱（LC–MS/MS）分析前实现高效且一致的样本处理。我们将该装置应用于间歇性禁食临床试验的人类血浆分析。本项纵向研究采用了为期8周的间歇性禁食干预，我们鉴定出了由该干预诱导的显著丰度变化，包括载脂蛋白A4（apolipoprotein A4, APOA4）水平升高，以及载脂蛋白C2（apolipoprotein C2, APOC2）与C3（apolipoprotein C3, APOC3）水平降低。这些变化与间歇性禁食干预后血浆甘油三酯的显著降低呈显著相关。鉴于这些蛋白质参与调控载脂蛋白颗粒代谢，我们提出间歇性禁食通过调节高密度脂蛋白（High-Density Lipoprotein, HDL）颗粒的大小与功能，对脂质代谢产生了积极影响。此外，我们采用了一款新型人类蛋白质变异数据库，以检测参与者体内的常见蛋白质变异。我们证实，能够稳定检测到源自多种蛋白质两个等位基因的临床相关肽段，其中包括一些与人类代谢表型相关的肽段。综上，这些研究结果证明了用于临床样本蛋白质组学分析的易用性工作流程的价值，可获得具有重要生物学意义的研究见解。