How does ProteinLynx Global Server pass 1 and 2 and modifications scoring work? - WKB1209

ENVIRONMENT

• ProteinLynx Global Server
• Results browser shows some peptides as "pass1" matches and others as "modifciation" or "pass 2" matches

ANSWER

Pass 1 (the first round of sequence matching) uses tighter tolerances and (by default) does not consider variable modifications. The result of pass 1 is a protein list that will be used in pass 2. This limits the number of matches we need to try in pass 2.

Pass 2 uses broader tolerances and looks for more variants, including variable modifications.

Scoring is the same for all peptides/proteins, regardless of whether they are found in pass 1 or pass 2. There is no bias against pass 2 matches, including those identified as carrying variable modifications.

For further details see Database searching and accounting of multiplexed precursor and product ion spectra from the data independent analysis of simple and complex peptide mixtures, Li et al, Proteomics 2009, 9, 1696–1719.

ADDITIONAL INFORMATION

If there are proteins in a sample that can only be found through matching the modified peptides, or if you are specifically interested in particular variable modifications, try marking them as "enriched" in the workflow parameters. Enriched variable modifications are searched in pass 1. Unfortunately, this tends to make IA slower and often hits more randoms more quickly. You can also try setting the false positive rate to 100%, to see all the matches that IA finds.