To better understand the acute inflammatory mechanisms, the modulation, and to investigate the key node in predicting inflammatory diseases, high-sensitivity LC-MS/MS-based proteomics and phosphoproteomics approaches were used to identify differential proteins in RAW264.7 macrophages with lipopolysaccharide (LPS). Furthermore, differential proteins and their main biological process, as well as signaling pathways, were analyzed through bioinformatics techniques. The biological process comparison revealed 219 differential proteins and 405 differential phosphorylation proteins, including major regulatory factors of metabolism (PFKL, PGK1, GYS1, ACC, HSL, LDHA, RAB14, PRKAA1), inflammatory signaling transduction (IKKs, NF-κB, IRAK, IKBkb, PI3K, AKT), and apoptosis (MCL-1, BID, NOXA, SQSTM1). Label-free proteome demonstrated canonical inflammation signaling pathways such as the TNF signaling pathway, NF-κB signaling pathway, and NOD-like receptor signaling pathway. Meanwhile, phosphoproteome revealed new areas of acute inflammation. Phosphoproteomics profiled that glycolysis was enhanced and lipid synthesis was increased. Overall, the AMPK signaling pathway is the key regulatory part in macrophages. These revealed that the early initiation phase of acute inflammation primarily regulated the phosphoproteins of glucose metabolic pathway and lipid synthesis to generate energy and molecules, along with the enhancement of pro-inflammatory factors, and further induced apoptosis. Phosphoproteomics provides new evidence for a complex network of specific but synergistically acting mechanisms confirming that metabolism has a key role in acute inflammation.
Keywords: acute inflammation; metabolism; phosphoproteomics; proteomics.