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.

由-/-PMM1和+/+PMM1样本来源的分泌物和总细胞裂解物通过基于稳定同位素二甲基标记的质谱定量蛋白质组学进行了比较。对分泌物和总细胞裂解物均进行了两次生物重复分析，其中后者已预先分为14个组分。具体而言，当肽属于-/-PMM1和+/+PMM1样本时，分别被标记为“轻”和“重”。因此，重/轻（H/L）比值大于1表明肽（进而，蛋白质）在-/-PMM1样本（轻）相对于+/+PMM1样本（重）中匮乏的程度，反之亦然。通过MaxQuant软件进行生物信息学分析，以鉴定和量化蛋白质组的变化。当在两次生物重复分析中均存在时，蛋白质比值高于1.5且低于0.5被视为由于PMM1敲除引起的蛋白质组可靠变化。