Comprehensive Analysis of the Proteome and PTMomes of C2C12 Myoblasts Reveals that Sialylation Plays a Role in the Differentiation of Skeletal Muscle Cells

The C2C12 myoblast is a model that
has been used extensively to
study the process of skeletal muscle differentiation. Proteomics has
advanced our understanding of skeletal muscle biology and also the
differentiation process of skeletal muscle cells. However, there is
still no comprehensive analysis of C2C12 myoblast proteomes, which
is important for the understanding of key drivers for the differentiation
of skeletal muscle cells. Here, we conducted multidimensional proteome
profiling to get a comprehensive analysis of proteomes and PTMomes
of C2C12 myoblasts with a TiSH strategy. A total of 8313 protein groups
were identified, including 7827 protein groups from nonmodified peptides,
3803 phosphoproteins, and 977 formerly sialylated N-linked glycoproteins.
Integrated analysis of proteomic and PTMomic data showed that almost
all of the kinases and transcription factors in the muscle cell differentiation
pathway were phosphorylated. Further analysis indicated that sialylation
might play a role in the differentiation of C2C12 myoblasts. Further
functional analysis demonstrated that C2C12 myoblasts showed a decreased
level of sialylation during skeletal muscle cell differentiation.
Inhibition of sialylation with the sialyltransferase inhibitor 3Fax-Neu5Ac
resulted in the lower expression of MHC and suppression of myoblast
fusion. In all, these results indicate that sialylation has an effect
on the differentiation of skeletal muscle cells.

C2C12肌原细胞模型已被广泛用于骨骼肌分化过程的研穷。蛋白质组学的研究深化了我们对骨骼肌生物学及其细胞分化进程的理解。然而，针对C2C12肌原细胞蛋白质组的综合分析尚付阙如，这对于揭示骨骼肌细胞分化关键驱动力至关重要。本研究采用TiSH策略，对C2C12肌原细胞的蛋白质组学和翻译后修饰组学进行了多维度的蛋白质组分析，共鉴定出8313个蛋白质组，其中包括7827个非修饰肽段蛋白质组、3803个磷酸化蛋白和977个曾发生唾液酸化的N-连接糖蛋白。蛋白质组学与翻译后修饰组学数据的综合分析显示，骨骼肌细胞分化途径中的几乎所有激酶和转录因子均发生了磷酸化。进一步分析表明，唾液酸化可能在C2C12肌原细胞的分化中发挥重要作用。进一步的功能分析证实，C2C12肌原细胞在骨骼肌细胞分化过程中唾液酸化水平降低。使用唾液酸转移酶抑制剂3Fax-Neu5Ac抑制唾液酸化，导致MHC表达降低和肌原细胞融合抑制。综上所述，这些结果表明唾液酸化对骨骼肌细胞的分化具有显著影响。