Identifying Proteins in a Top Down Standard with ProSightPD 4.0.

A simple mixture of five proteins is used by the National Resource for Translational and Developmental Proteomics to check the health of their mass spectrometers prior to running clinical samples.

The Top Down Standards mixture consists of the following proteins:

  • 1 pmol myoglobin (M5696)
  • 0.5 pmol trypsinogen (T1143)
  • 0.5 pmol carbonic anhydrase (C2624)
  • 0.1 pmol ubiquitin (U6253)
  • Superoxide dismutase – SOD1 – contaminant is present in carbonic anhydrase

An example RAW file containing data of Top Down Standard proteins can be downloaded and searched against a custom ProSightPC database to identify proteins in the standard’s mix.

A search can also be performed in ProSightPD as a quick exercise to check that ProSightPD is working correctly on your computer prior to attempting analyses of your own files.

Following these steps to do this analysis:

1. Ensure that both Proteome Discoverer 2.5 and ProSightPD 4.0 are installed on the computer on which you intend to run this exercise. If Proteome Discoverer 2.5 or ProSightPD 4.0 is not installed, their installers can be found on the Thermo’s Life Sciences Mass Spectrometry Software Download and Licensing Portal here.

2. Download the Standards .raw file and the Top Down Standards database (.psdb), and the Top Down Standards Analysis template.

3. Follow the steps in this video to install the Proteoform Database for Top Down Standards, and run the search.

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