1. Select a search engine (e.g. ProLuCID), then the following screen should appear.

Figure 9.1.1: ProLuCID search

2. Select the location for the computational search to be performed, either “Cluster” or “Cloud,” depending on your available resources.

3. Make the appropriate selections within the “Basic parameters” section:

  • Select a “Protein database” for the organism from which your sample originated. For this example analysis, mouse was the sample origin so we select a mouse database. See the DATABASE manual for uploading databases if one is not present for your sample organism.
  • Select the “Fragmentation/activation method” for which MS/MS spectra were acquired. For this example analysis, multistage-activation CID was used so we selected “CID.”
  • Select the “Precursor/peptide mass tolerance”. An Orbitrap was used for these MS analyses, so we select “High resolution”. Although it is not as high confidence for phosphorylation analysis, if low resolution precursor data was acquired (i.e. LTQ), select “Low resolution”. Please see Section 4 of this manual for a description of the precursor and fragment mass tolerance default values.

4. Choose the appropriate options within the “Enzyme specificity” section. Trypsin was used for this sample, but see Section 4 of this manual for a description of settings for other proteases:

  • Select a “Specificity” of “one end” and a “Max num internal miscleavage” of “unlimited”. Phosphorylation events near lysine or arginine residues can inhibit trypsin cleavage, so leaving the option for only one tryptic end and more than 3 missed cleavages is advantageous to identifying more phosphopeptides. If adequate computational resources are available, a “Specificity” of “none” can be used to potentially find all phosphopeptides.
  • Leave the defaults of “Protease name” of “trypsin,” “Residues” of “KR,” and “Cut position” of “C-term.”

5. If cysteine residues were carbamidomethylated with iodoacetamide or chloroacetamide, then “57.02146 C” should be added in the “Amino acid specific static modifications” box.

6. Continue adding search parameters in the “Differential/variable modifications” section as shown below:

  • Enter the number of phosphorylation modifications expected or desired to find per phosphopeptide; 3 is commonly used for most phosphopeptide enrichment methods.
  • Add the mass and residues (79.9663 STY) for differential phosphorylation on serine, threonine, and tyrosine in the “Differential Modification” box.
  • If the experiment was quantitative (i.e. 15N or SILAC), select the appropriate “Metabolic Labeling Search” options. For this example, we selected “Yes” and “N15”. If SILAC was used, select “Selected Amino Acids” and enter the appropriate mass shifts. A full description for this can be found in Section 8 of this manual.
  • Unique to phosphorylation MS/MS analysis, multistage activation CID fragmentation can be used to improve the fragmentation of peptides after neutral loss of phosphate. [MSA ref] If this method was used, select “Multistage activation mode,” option “1” to search for both normal and neutral loss fragment ions, and option “2” to search for only neutral loss fragment ions.

Figure 9.1.2: Differential/variable modifications

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