The inducible and reversible modification of proteins with polyubiquitin chains has emerged as one of the most common and versatile means of protein modification. Ubiquitin contains 7 lysine residues, each of which can serve as an acceptor site for formation of polyubiquitin chains. In addition, the C-terminal glycine of one ubiquitin molecule can form a conventional peptide bond with the N-terminal methionine of an acceptor ubiquitin, leading to the formation of linear polyubiquitin chains. Linear ubiquitin chains stabilize proteins and play critical roles in signaling complex assembly. The linear ubiquitin chain assembly complex (LUBAC) consists of at least 3 proteins: HOIP (also known as RNF31), HOIL-1L (also known as RBCK1), and SHARPIN [1-6]. LUBAC is the only E3 ligase known to assemble linear polyubiquitin chains. Importantly, LUBAC was shown to target components of the canonical NF-κB signaling pathway, which is vital for development of inflammation and immunity [2, 5]. Our understanding of linear ubiquitination primarily comes from studies involving TNFα signaling. Upon stimulation with the cytokine TNFα, LUBAC is recruited to the TNFα receptor 1 (TNFR1)[1, 5]. LUBAC regulates TNFα signaling by targeting the regulatory protein, NF-κB essential modulator (NEMO), for nondegradative linear ubiquitination. Depletion of LUBAC components by RNA interference or genetic ablation attenuates cytokine-driven NF-κB signaling [1-5, 7]. LUBAC is a 600 kDa multimeric complex [6, 8, 9]. However, the stoichiometry and additional components associated with the LUBAC protein complex remain unknown. We used affinity protein purification in HEK293 cells stably expressing Flag-tagged HOIL-1L, HOIP and SHARPIN, followed by mass spectrometry to identify proteins that interact with LUBAC and contribute to its function. For data processing, we compared the affinity purification coupled with mass spectrometry (AP-MS) data of LUBAC with our laboratory database containing 208 protein complexes from HEK293 cells. Our database analysis tool, termed ZSPORE, is designed to identify high confidence interacting proteins (HCIPs) and filter out nonspecific binding proteins (NSBP) from AP-MS datasets [10]. ZSPORE integrates three parameters: z-score based on total spectral counts (z-score> 5; P< 0.0001), prey occurrence (< 5%, as a measure of uniqueness) and reproducibility (≥ 50% in 4 runs). In total, 32 HCIPs were uncovered (Supplementary information, Table S1). Reciprocal interactions among HOIL-1L, HOIP and SHARPIN were detected, indicating the strong associations among these three LUBAC components. In addition, HOIL-1L, HOIP and SHARPIN formed individual subnetworks with unique partners. Several known interactors, such as TRAF2, BIRC2 (also known as cIAP1), FAM105B (also termed OTULIN) and DZIP3, were present in the LUBAC interactome (Supplementary information, Figure S1A). However, 70% of the interactions were not found in literature searches or the STRING and BioGRID databases (Supplementary information, Figure S1A). Gene Ontology (GO) term enrichment analysis indicates that 41% of HCIPs are involved in the ubiquitination process, including several E3 ligases, deubiquitinases and one ubiquitin conjugating E2 enzyme (UBE2L3, also known as UbcH7 or UbcM4)(Supplementary information, Figure S1A). To determine whether these protein associations were dependent on TNFα stimulation, we compared the ratio of total spectral counts with or without TNFα treatment. Under the experimental conditions tested, TNFα treatment did not induce> 5 fold changes in normalized spectral counts …