Beneficial effects of Glc-1,6-P2 modulation on mutant phosphomannomutase-2_Proteomics

Secretomes and total cell lysates deriving from -/-PMM1 and +/+PMM1 samples were compared by mass spectrometry-based quantitative proteomic through Stable Isotope Dimethyl Labeling. Two biological replicates were analysed for both secretomes and total cell lysates, with the latter being pre-fractionated in 14 fractions.
More in details, peptides were labeled as “light” and “heavy” when belonging to -/-PMM1 and +/+PMM1 samples, respectively. Thus, a heavy/light (H/L) ratio ˃ 1 indicates how much a peptide (and consequently, a protein) is scarce in the -/-PMM1 sample (light) in respect to the +/+PMM1 (heavy) one and vice versa. Bioinformatic analysis was performed through MaxQuant software to identify and quantify proteome alterations. Protein ratios higher than 1.5 and lower than 0.5 were considered reliable alterations of the proteome due to PMM1 knock-out, when existing in both biological replicates.

本研究采用稳定同位素二甲基标记（Stable Isotope Dimethyl Labeling）结合基于质谱的定量蛋白质组学方法，对PMM1基因纯合敲除（-/-PMM1）与野生型（+/+PMM1）样本的分泌组（secretomes）及全细胞裂解液（total cell lysates）进行了对比分析。分泌组与全细胞裂解液样本均设置2次生物学重复，其中全细胞裂解液预先被分为14个组分完成分级分离。
具体而言，对应-/-PMM1样本的肽段被标记为"轻量（light）"，对应+/+PMM1样本的肽段则被标记为"重量（heavy）"。由此，重量/轻量（H/L）比值大于1时，代表该肽段（及其对应的蛋白质）在-/-PMM1样本（轻量标记组）中的丰度低于+/+PMM1样本（重量标记组），反之亦然。本研究借助MaxQuant软件开展生物信息学分析，以鉴定并定量蛋白质组的变化差异。当蛋白质的丰度比值大于1.5或小于0.5，且该变化在两次生物学重复中均存在时，将其认定为PMM1基因敲除引发的可靠蛋白质组差异。