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Updated: Aug 8, 2026
Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling
Published on: April 1, 2017
High-throughput analysis of protein/peptide complexes by immunoprecipitation and automated LC-MS/MS
Zhaosheng Lin1, David K Crockett, Megan S Lim
1ARUP Institute for Clinical and Experimental Pathology, University of Utah Health Science Center, 50 North Medical Drive, Salt Lake City, Utah 84132, USA.
Insights
We developed a high-throughput nanoflow LC-MS/MS method for analyzing protein complexes. This automated technique efficiently identifies interacting proteins, aiding in understanding cell signaling and discovering disease markers.
Area of Science:
- Proteomics
- Cell Signaling
- Biochemistry
Background:
- Identifying protein interactions is crucial for understanding cell signaling pathways and discovering disease markers.
- Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a common method for analyzing protein complexes.
- Existing LC-MS/MS methods face challenges with large dwell volumes in high-throughput analysis.
Purpose of the Study:
- To develop a high-throughput nanoflow LC-MS/MS method for sensitive analysis of protein/peptide complexes.
- To overcome limitations of conventional autosampler setups in microcapillary LC-MS/MS.
- To demonstrate the utility of the method for identifying proteins interacting with p38 MAP kinase.
Main Methods:
- Employed a valve-controlled variable flow method with a peptide trap to manage autosampler dwell volume.
- Utilized a microcapillary C18 column for peptide separation and an ion-trap MS/MS for analysis.
- Applied the automated LC-MS/MS method to analyze peptides from immunoprecipitated p38 MAP kinase complexes.
Main Results:
- Successfully analyzed over 40 protein/peptide samples at femtomole levels continuously.
- Identified more than 50 proteins from 37 excised gel bands, including cytoskeletal, ribosomal, and signaling proteins.
- Demonstrated the method's sensitivity and high-throughput capability for complex proteomic analysis.
Conclusions:
- The automated nanoflow LC-MS/MS method is effective for sensitive and high-throughput analysis of protein/peptide complexes.
- This approach facilitates peptide interaction mapping and other proteomic studies.
- The identified proteins are potential interactors of p38 MAP kinase, offering insights into signaling pathways.
Background:
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.
Approach:
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.
Results And Conclusions:
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.
