Antibody validation using ip-mass spectrometry
A mass spectrometry and antibody technology, which can be used in material testing products, instruments, measuring devices, etc., and can solve the problems of difficulty in filtering and interpreting mass spectrometry results.
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Embodiment 1
[0120] Example 1. Selection of cell models and targets for IP-MS antibody validation
[0121] figure 1 A summary of the IP-MS antibody validation process is presented. First, protein targets and antibodies are selected for validation. An appropriate cell model believed to contain the protein target of interest is selected, cultured, and lysates prepared. A "deep dive" analysis of many different cell lines was performed to select an appropriate cell line for use with our initial IP-MS analysis. Target proteins are immunoenriched from cell lysates using antibodies believed to be specific for a certain target protein (eg via IP / Western blotting, but not verified via IP-MS). Following IP, samples were analyzed by MS and novel bioinformatics analyzes were performed. Qualitative and quantitative data pertaining to each antibody's ability to bind the target protein is captured by this process, as well as data pertaining to non-target interacting proteins, as well as non-specific ...
Embodiment 2
[0141] Embodiment 2.IP / MS scheme
[0142] Use the following method to use figure 1 The IP / MS protocol shown generated data in a number of different cell lines.
[0143] Antibodies against TP53, CDH1, CDH2 and CDKN1A were purchased from Thermo Fisher Scientific (ref. Figure 5A , 5B , 7A). Antibodies were screened and validated using the Thermo Scientific Pierce MS-Compatible Magnetic IP Kit (Protein A / G) (Thermo Fisher Scientific, PN: 90409) as described in the instruction manual. 500 μg of lysate and 5 μg of antibody were used for all experiments. The IP eluate was dried in a vacuum concentrator and the sample was processed by digestion in solution as recommended in the instruction manual (Thermo Fisher Scientific, PN: 90409). Dried digested samples were resuspended in 13 μL of 4% acetonitrile and 0.2% formic acid and transferred into autosampler vials followed by LC-MS analysis.
[0144] LC-MS Analysis of IP-MS Samples
[0145] Use with THERMO TM SCIENTIFIC TM Q EXA...
Embodiment 3
[0147] Example 3. Identification of targets by MS
[0148] Validation of antibodies was assisted using protein expression profiling from Example 1 and techniques from Example 2 using a protocol combining immunoprecipitation with mass spectrometry (IP-MS). A key benefit of antibody validation by IP-MS is the identification of the native target protein and its isoforms and modifications. As mentioned above, the MS results of this target identification can be assessed in several ways, including the number of unique peptides, protein sequence coverage, the number of spectra observed for peptides from the target protein (spectrum counts) or from detected peptides. Integrated MS signal intensities of a subset or all of the peptides. Regardless of the measurement protocol, the relative performance of various antibodies on the same target can be readily compared. For example, immunoprecipitation using 13 IP / Western blot validated antibodies against the p53 protein (TP53) was assesse...
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