Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, California 90048, United States.
Advanced Clinical Biosystems Research Institute, Smidt Heart institute, Cedars-Sinai Medical Center, Los Angeles, California 90048, United States.
J Proteome Res. 2023 Jun 2;22(6):2124-2130. doi: 10.1021/acs.jproteome.3c00027. Epub 2023 Apr 11.
Heart tissue sample preparation for mass spectrometry (MS) analysis that includes prefractionation reduces the cellular protein dynamic range and increases the relative abundance of nonsarcomeric proteins. We previously described "IN-Sequence" (IN-Seq) where heart tissue lysate is sequentially partitioned into three subcellular fractions to increase the proteome coverage more than a single direct tissue analysis by mass spectrometry. Here, we report an adaptation of the high-field asymmetric ion mobility spectrometry (FAIMS) coupled to mass spectrometry, and the establishment of a simple one step sample preparation coupled with gas-phase fractionation. The FAIMS approach substantially reduces manual sample handling, significantly shortens the MS instrument processing time, and produces unique protein identification and quantification approximating the commonly used IN-Seq method in less time.
用于质谱 (MS) 分析的心脏组织样品制备，包括预分级，会降低细胞蛋白质动态范围并增加非肌节蛋白的相对丰度。我们之前描述了“IN-序列”（IN-Seq），其中心脏组织裂解物依次分为三个亚细胞部分，通过 MS 比单次直接组织分析增加了更多的蛋白质组覆盖范围。在这里，我们报告了一种高场非对称离子淌度谱 (FAIMS) 与质谱的适应方法，以及与气相分级相结合的简单一步样品制备方法的建立。FAIMS 方法大大减少了手动样品处理，显著缩短了 MS 仪器处理时间，并在更短的时间内产生了独特的蛋白质鉴定和定量，类似于常用的 IN-Seq 方法。
