Optimizing Workflows for LC/MS Analysis of Co-Immunoprecipitated Protein Complexes - "Soap Opera(tions)".

The ability to define specific, and often dynamic proteinprotein associations within interactomes is critical to advancing an understanding of cell signal transduction pathways, drug design and the actions of therapeutic agents [1]. Standard approaches to assessing protein-protein interactions commonly have involved the retrieval of target proteins (“bait”) plus partner protein coimmunoprecipitates; the isolation of partner proteins from 1D or 2D gels; and the identification of retrieved partners via LC-MS/MS [2]. The use of in vivo biotinylation [3,4] or HALO tagging of target / bait proteins [5,6] can further extend the detection of protein partnerships [7]. Limitations, however, can include the need for multi-step workflows, relatively large-scale preparations, and/or the construction of labeled baits. Shotgun approaches including MudPIT (multidimensional protein identification technologies) [8] also continue to be advanced that can bypass SDS-PAGE, and in certain formats avoid the need for elution of immune complexes from adsorbent gels. In RIME (Rapid Immunoprecipitation Mass Spectrometry) co-IP LC/MS, for example, immune complexes are proteolyzed from Ig or Igprotein A/G/L beads and are analyzed directly by LC/MS [9,10]. Beyond this, in silico procedures also have recently been developed to guide cell lysis and/or subcellular fraction extraction methods, and co-IP workflows [11,12]. For LCMS/MS data analysis, advanced algorithms and reference databases also continue to be developed (e.g., DIA/SWATH) [13]. During the earlier fundamental steps of cell lysate preparation and target/ bait plus partner protein immunoprecipitation, the choice of detergents also becomes a point of central importance. This relates to the need to solubilize, but not disrupt protein complexes, while avoiding detergent incompatibilities with LC/MS. In a context of cell signal transduction, membrane proteins additionally can be key components (including transmembrane receptors), and this brings further attention to detergent considerations [14]. In particular, this includes a requirement to retain the solubility of hydrophobic proteins, while also maintaining protein-protein partnerships [15,16]. One non-ionic detergent frequently suggested as a potentially advantageous choice for this challenge is octyl beta-D-glucoside (OBG). This is based on OBG’s effectiveness in solubilizing and retrieving membrane proteins [17,18], and OBG’s exceptional property of rapid micelle disassembly upon dilution or dialysis [17,19]. In published studies, however, comparably few examples exist for OBG’s use in cell lysis and co-IP (compared, for example, with Triton-X-100, Igepal, DOC, CHAPS). This includes systems that use FLAG-epitope tagged target/bait proteins. In a workflow context, a challenge also exists during immune complex isolation for replacing OBG (an LC-MS incompatible detergent) with an LC-MS compatible detergent that continues to preserve solubilized co-IP complexes. For LC-MS compatible detergents, new options are emerging [11,16,20]. Most continue to be ionic and/or strong detergents, several of which are proving to be useful Optimizing Workflows for LC/MS Analysis of Co-Immunoprecipitated Protein Complexes – “Soap Opera (tions)”