We developed a rapid method for multiplex substrate profiling to reveal the extended substrate specificity of post translational modifying enzymes using liquid chromatography-tandem mass spectrometry sequencing. The methodology is referred to as Multiplex Substrate Profiling by Mass Spectrometry (MSPMS) and the initial publication is PMC3707110 (Global Identification of Peptidase Specificity by Multiplex Substrate Profiling). Analysis of MSP-MS samples is facilitated with the MSP-xtractor software that extracts the peptide cleavage site and spectral counts of the corresponding cleavage products. Spectral counts are used for the relative quantification of peptide cleavage products.
Extractor is a software created by UCSF to extract cleavage data from peak lists generated from mass spectrometry data of MSP-MS experiments. Raw MS data files are usually converted to peak lists text files (.*txt) by Protein Prospector, where files are batch-tagged against a list of all possible cleavages observed in the MSP-MS peptide library.
When multiple timepoints are employed in a MSP-MS assay, raw MS files generated for individual timepoints can be combined by Protein Prospector to a single text file. Extractor is the software that can extract cleavage data from each time-point and give researchers a glimpse of cleavage kinetics for each individual peptide from the MSP-MS library.
- Microsoft Windows 10
- Microsoft Visual C++ 2015
- 8 GB RAM
How to use
- When you open Extractor, you will be prompted to “Please select the oligopeptide library file you want to use as a reference.”
- For MSP-MS analysis, import the file “TDP_237_library_Met.txt.”
- Next you will be prompted to “Please select the Report Search Compare file that you want to use as data.”
- You should import your text file generated by Prospector as a peaklist of combined results from your different MSP-MS timepoints.
- If your file was assembled correctly, you will receive a “Done” green message. If not, a red message will appear, suggesting that you should review your file.
- To comply with MSP-MS standards, keep both “Desired Left/Right Padding from Cleavage” as 4. This will yield observed cleavaged results as a P4-P4' peptide sequences.
- Choose if you want Longform Results (for a full report of analysis) or Summary Results (for a short one).
- The resulting text files (*.txt) can be opened in Excel, R, or your preferred text/table manager.