High-throughput analysis of protein/peptide complexes by immunoprecipitation and automated LC-MS/MS

The identification of interacting proteins within protein complexes is key to understanding the transduction and regulation of cell signaling pathways, and is also a useful tool for identifying novel disease markers. Immunoprecipitation of protein complexes followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been used to identify targets that bind to a protein of interest. Here, we report a high-throughput nanoflow LC-MS/MS method for analysis of protein/peptide complexes.

To overcome the large dwell volume from connecting lines and the injector when using an autosampler in microcapillary LC-MS/MS, we employed a valve-controlled variable flow method with a peptide trap. This method enables fast trapping of peptides from samples injected by an autosampler within minutes followed by a split-controlled nanoflow of acetonitrile gradient through a microcapillary C18 column to separate and elute peptides into the ion-trap MS/MS. Over 40 protein/peptide samples at femtomole levels could be analyzed continuously using the same in-house packed microcapillary C18 column. The p38 mitogen-activated protein kinase (p38 MAP kinase) is a signaling protein that is involved in transduction of extracellular signals including oxidative stress, growth factors, and cytokines, with diverse cellular consequences such as cell proliferation, differentiation, or apoptosis. Immunocomplex of monoclonal anti-p38 beta antibodies from 2 mg total lysates from the OCI LY-1 lymphoma cell line was resolved in 1D-PAGE gel followed by silver staining of the gel. Protein bands were excised and digested with trypsin. Peptides were extracted and analyzed using the automated LC-MS/MS method.

From 37 excised protein bands in the 1D-PAGE gel, we identified more than 50 proteins, including cytoskeletal proteins, ribosomal proteins, transcription factors, and KIAA potential signaling proteins. These proteins are the potential targets that may interact directly or indirectly with the p38 MAP kinase proteins. Our studies demonstrate the utility of automated nanoflow LC-MS/MS for sensitive and high-throughput analysis of protein/peptide complexes. Utilization of methods based on this principle would greatly facilitate peptide interaction mapping and other proteomic studies requiring high-throughput pre-analytical sample preparation.