骨肉瘤（OS）是最常见的骨原发性恶性肿瘤，占所有骨癌的20%。为了增加对骨肉瘤生物学的理解，我们开发并评估了一种自上而下的质谱法来检测、鉴定和定量骨肉瘤细胞中低分子量（即1千道尔顿 < 分子量 < 30千道尔顿）的蛋白质。使用反相纳米液相色谱和高分辨质谱技术获得了自上而下蛋白质组数据，结果确定了328个蛋白质和820个保守型或降解产物的高置信度。本研究鉴定了8种翻译后修饰（PTMs），包括N-末端乙酰化、赖氨酸乙酰化、琥珀酰化、丙酰化、丝氨酸/酪氨酸磷酸化、组氨酸甲基化和N-乙酰亮氨酸。我们证实，一个截短的N-末端保守型通过去除N-末端Met（-131道尔顿），第二个氨基酸的乙酰化（+42道尔顿）和Met氧化（+16道尔顿）失去了73道尔顿的质量。结果表明，保守型/可降解肽的水平与骨肉瘤细胞系的转移表型相关。该研究展示了自上而下蛋白质组学在骨肉瘤生物学的特征和相对定量上的好处，以及小分子量保守型作为仍未被充分重视的生物标志物来源的潜力。

Osteosarcoma (OS) is the most common primary malignant tumor of bone, which occupying about 20% of all bone cancers. To increase understanding of the biology of OS, we developed and evaluated a top-down mass spectrometry approach to detect, identify and quantify low molecular weight (MW) proteins (i.e., 1 kDa < MW < 30 kDa) in osteosarcoma cells. Top-down proteomic (TDP) data was acquired using reversed phase nano-liquid chromatography in conjunction with high-resolution mass spectrometry and resulted in the assignment of 328 proteins and 820 proteoforms or degradation products with high confidence. Eight post-translational modifications (PTMs) were identified in the present study, including N-terminal acetylation, lysine acetylation, succinylation, malonylation, serine/tyrosine phosphorylation, histidine methylation and N-acetylleucine. We confirmed that a truncated N-terminal proteoform lost 73 Da of mass through removal of the N-terminal Met (-131 Da), acetylation of the second amino acid (+42 Da), and Met oxidation (+16 Da). The results showed that the levels of proteoforms/biodegradable peptides correlated with the metastatic phenotypes of osteosarcoma cell lines. This study demonstrates the benefits of TDP for the characterization and relative quantification of proteoforms with relevance to OS biology and the potential of small molecular weight proteoforms to serve as a still underappreciated source of biomarkers.