Direct Proteomic Quantification of the Secretome of Activated Immune Cells

Tracking Secreted Proteins The proteins secreted by cells provide a flow of information within tissues and are thus of particular interest. However, systematic detection of secreted proteins is tricky because they tend to be present in small amounts within complex mixtures of proteins where other components are very abundant. Meissner et al. (p. 475) developed a method for screening the secreted proteins from cultured mouse macrophages in response to cues that cause inflammation. The amount of contaminating proteins was reduced by culturing the cells without added serum and then sensitive mass spectrometry techniques were used to detect and quantify secretion of nearly 800 different proteins. Secretion was compared from cells lacking the signaling adaptor proteins MyD88 or TRIF, or both. Secretion of some proteins were regulated redundantly and were secreted without one of the adaptors, but others required both signals for release. Some anti-inflammatory proteins were released at later times in response to synergistic signals from both adaptor proteins, perhaps as a fail-safe mechanism to prevent excessive inflammation. A mass spectrometry–based method detects picogram quantities of proteins secreted by macrophages. Protein secretion allows communication of distant cells in an organism and controls a broad range of physiological functions. We describe a quantitative, high-resolution mass spectrometric workflow to detect and quantify proteins that are released from immune cells upon receptor ligation. We quantified the time-resolved release of 775 proteins, including 52 annotated cytokines from only 150,000 primary Toll-like receptor 4–activated macrophages per condition. Achieving low picogram sensitivity, we detected secreted proteins whose abundance increased by a factor of more than 10,000 upon stimulation. Secretome to transcriptome comparisons revealed the transcriptionally decoupled release of lysosomal proteins. From genetic models, we defined secretory profiles that depended on distinct intracellular signaling adaptors and showed that secretion of many proinflammatory proteins is safeguarded by redundant mechanisms, whereas signaling adaptor synergy promoted the release of anti-inflammatory proteins.