Mass spectrometry identified Ser-316 phosphorylation of p65 in response to IL-1β. Tandem mass spectrometry (ms2) of precursor ions in the phosphorylated p65 peptide (amino acids 316–329; sequence SPFSGPTDPRPPPR, where S (red) indicates phosphorylated serine). Black lines indicate peptide cleavage. Compared with unmodified 316–329 peptide, an 80-Da mass shift was observed on precursor ion and b series ions such as b3, b7, and b8, but not on y series ions from y4 to y13, indicating phosphorylation of Ser-316.