i) Select phosphosites (manual)

This is the most detailed and flexible option for assay design. It allows for manual selection of each phosphopeptide sequence and charge state to be measured and direct inspection of representative MS/MS spectra. It is the best way to directly evaluate data supporting an individual phosphorylation site. Targeting multiple sequences and charge states provides the best chances of detecting the site.

1. Start typing a protein identifier (HGNC gene symbol or Uniprot/Swiss-Prot accession are supported) and choose from the drop-down list.

   

2. Select the site (position) on the protein. All peptide species that have been observed for this phosphosite will populate the table. Check the box next to the peptide(s) you would like to add or select all using the box in the top right. The most frequently observed sequence is selected by default. Choose “Select all positional isomers” to automatically schedule other known phosphosites that are localized to the same peptide (e.g. THFPQFS[80]YSASIRE and THFPQFSYSAS[80]IRE).

   

3. Choose “Select Peptides”. A list of precursor ions that have been observed for each peptide should appear.

   

4. Click on the peptide sequence to see its MS/MS spectrum.

   

   MS/MS spectra are visualized using Lorikeet (http://uwpr.github.io/Lorikeet/)

   

5. Check the box next to the precursor you would like to add or top right box to select all. The most frequently observed charge state is selected by default. Click “Add selected peptides”. The selected phosphopeptide precursor ions will be added to the current assay schedule which can be visualized at the bottom of the page (See section entitled “Assay Refinement”)

   

6. Repeat steps 1-5 to add more targets.

ii) Select phosphosites (automatic)

This option is similar as above, but precursor selection is performed automatically. It is the fastest way to browse the database to add individual phosphosites to an assay.

1. Start typing a protein identifier (HGNC gene symbol or Uniprot/Swiss-Prot accession are supported) and choose from the drop-down list. All phosphorylation site isoforms that have been observed on the protein will populate the table.

2. Check the boxes next to the phosphorylation sites you would like to add to the current assay or the top right box to select all.

   

3. Click “Add Selected Sites”. The most frequently observed peptide sequence and charge state will be automatically added to the current assay schedule which can be visualized at the bottom of the page (See section entitled “Assay refinement”)

   

iii) Browse pre-built assays

This option provides a menu of pre-built lists of phosphorylation sites curated from phosphospecific antibody catalogs, pathway diagrams, or derived on the basis of protein structural features. It is the easiest way to assemble a large assay containing phosphosites of known function. These lists are provided as a starting point to simplify assay design. They are not comprehensive nor are they necessarily generalizable to the unique signaling biology of specific human cell types.

1. Select a pathway from the drop-down menu. Check the boxes next to the phosphorylation sites you would like to add to the current assay or top right box to select all.

   

2. Click “Add Selected Sites”. The most frequently observed peptide sequence and charge state will be automatically added to the current assay schedule which can be visualized at the bottom of the page (See section entitled “Assay Refinement”)

   

3. Add more phosphosites from other pathways and proteins to create a customized assay for your experiment.

iv) Upload a list of sites

This option allows you to upload a custom list of phosphorylation sites for automatic precursor selection. It is the fastest way to generate a large assay for example to validate or expand on results from discovery experiments.

1. Click “Upload file” to generate a dialog for .csv file upload. The file must be in the format shown below, using HGNC gene symbols or Uniprot ID.

   

2. Check the boxes next to the phosphorylation sites you would like to add to the current assay or the top right box to select all.

   

3. Click “Add Selected Sites”. The most frequently observed peptide sequence and charge state will be automatically added to the current assay schedule which can be visualized at the bottom of the page (See section entitled “Refine assay”)

   

i) Pierce™ PRTC mixture (cat#. 88320)

1. Analyze the PRTC peptide mixture using your LC-MS/MS system.

   

2. Enter the empirical retention times of each peptide directly in the table as below OR download the template .csv file, edit the table, and re-upload.

   

3. Enter the total length of the method in minutes in the box labeled “Run time” and click “Calibrate”. The retention times of the current assay will be updated automatically.

   

ii) Bovine serum albumin (BSA) digest

1. Prepare a BSA peptide standard by digesting BSA protein using trypsin. Analyze the BSA peptide mixture using your LC-MS/MS system. Some representative peptides are illustrated below, but more than 100 BSA-derived peptides are included in the retention time database. For this reason we recommend analyzing the sample using DDA to identify as many peptides as possible.

   

2. Enter the empirical retention times of each peptide directly in the table as below (not recommended) OR download the template .csv file and upload a complete list of BSA peptides identified using DDA (recommended).

   

3. Enter the total length of the method in minutes in the box labeled “Run time” and click “Calibrate”. The retention times of the current assay will be updated automatically.

   

iii) Phosphopeptide mixture (human)

1. Prepare an aliquot of phosphopeptides from a sample you will be using for your experiment or prepare a human phosphopeptide sample similar to those to be analyzed in your experiment (e.g. use a HeLa lysate if your experiment is performed in HeLa cells). Analyze by DDA using your LC-MS/MS system.

2. Upload the peptide sequences and retention times using the template provided.

   

3. Enter the total length of the method in minutes in the box labeled “Run time” and click “Calibrate”. The retention times of the current assay will be updated automatically.

   

Using the portal:

1. Enter the desired retention time tolerance window in the box. This represents the length of time before and after the predicted retention time to monitor for each phosphopeptide. The default tolerance is +/- 5 minutes, which was sufficient to capture >99% of targets using DDA analysis of a phosphopeptide mixture for calibration. Click refresh to update the schedule density plot.

   

2. [Optional] Continue to add or remove phosphopeptide precursors to reach the desired number of concurrent precursors (precursor density). Click the (x) next to a peptide to remove it from the assay.

   

3. Click “Upload schedule” to upload an assay previously generated using this tool. This is helpful for adding new targets and recalibrating the retention times of old assays.

   

4. Click “Merge positional isomers” (e.g. THFPQFS[80]YSASIRE and THFPQFSYSAS[80]IRE) to merge peptides with the same precursor m/z. Since these precursors often have overlapping windows, this eliminates redundant sampling.