Ying Guojin, He Yu, Yang Mengqing, Lu Gang, Li Yang, Cui Wei, Hu Zhengyan, Zhang Zhenbin
Institute of Drug Discovery Technology, Ningbo University, Ningbo, Zhejiang 315211, China.
Translational Medicine Center of Pain, Emotion and Cognition, Zhejiang Provincial Key Laboratory of Pathophysiology, Health Science Center, Ningbo University, Ningbo, Zhejiang 315211, China.
Anal Chem. 2025 Feb 4;97(4):2111-2119. doi: 10.1021/acs.analchem.4c04857. Epub 2025 Jan 24.
A rapid, sensitive, and high-throughput sample preparation method is of paramount significance for proteomics analysis. Here, we report a fast, high-sensitivity MICROFASP method that is capable of completing sample preparation within 1.5 h, enhancing the throughput by over 13 times compared to the previous reports. Protein digestion time was significantly cut from 17 h to 20 min in a limited volume. Simultaneous reduction and alkylation occurred within 30 min. The label-free quantitation intensities of proteins from the fast and conventional MICROFASP methods were highly correlated ( = 0.91), validating the reliability of the fast-MICROFASP method. When starting with 1 μg of K562 cell lysate, the fast-MICROFASP method identified over 6 times more protein groups and 19 times more peptides than did the iST method. A 96-well plate-based version was developed to process 8 brain tissue samples from APP/PS1 transgenic mice in parallel. Averagely, with only 1 μg of protein lysate, 2826 protein groups ( = 8, RSD = 0.7%) and 12,972 peptides ( = 8, RSD = 1.5%) were identified from each sample. Amyloid-beta protein was successfully identified as a highly expressed protein, which shows its potential for detecting diagnostic markers and proteome profiling with low-microgram samples. We anticipate the high-sensitivity 96-well plate-based fast-MICROFASP method will have wide application in high-throughput and rapid preparation of large cohorts of low-microgram samples (e.g., clinical biopsy) for comprehensive proteome profiling. Data are available via ProteomeXchange with the identifier PXD053720.
一种快速、灵敏且高通量的样品制备方法对于蛋白质组学分析至关重要。在此，我们报告了一种快速、高灵敏度的MICROFASP方法，该方法能够在1.5小时内完成样品制备，与先前的报告相比，通量提高了13倍以上。在有限体积内，蛋白质消化时间从17小时显著缩短至20分钟。同时还原和烷基化在30分钟内完成。快速和传统MICROFASP方法的蛋白质无标记定量强度高度相关（ = 0.91），验证了快速MICROFASP方法的可靠性。当以1μg K562细胞裂解物开始时，快速MICROFASP方法鉴定出的蛋白质组数量比iST方法多6倍以上，肽段数量多19倍。开发了一种基于96孔板的版本，用于并行处理来自APP/PS1转基因小鼠的8个脑组织样品。平均而言，仅用1μg蛋白质裂解物，每个样品就鉴定出2826个蛋白质组（ = 8，RSD = 0.7%）和12972个肽段（ = 8，RSD = 1.5%）。淀粉样β蛋白被成功鉴定为高表达蛋白，这表明其在检测低微克样品的诊断标志物和蛋白质组分析方面具有潜力。我们预计基于96孔板的高灵敏度快速MICROFASP方法将在高通量和快速制备大量低微克样品（如临床活检）以进行全面蛋白质组分析方面有广泛应用。数据可通过ProteomeXchange获得，标识符为PXD053720。
