We refer to “Skyline iRT Retention Time Prediction (Page 2-9)” and want to calibrate retention time of all experiment runs. First, we have created a document (named as peptide iRT) of retention time predictor (Edit iRT calculator) using enolase digest peptides (from cell lysate digest experiment) that span the retention-time range of we're interested.

Save document as .sky file.

Next, we open a new file, on the peptide settings  Retention time predictor  selected peptide iRT .

Configuring Peptide Setting and Transition Settings Importing a FASTA file Save as new .sky file Importing raw date (results). Next, we have performed the step of “adding iRT values for new targeted peptides” (refer to “Skyline iRT Retention Time Prediction (Page 8-9)) . After doing above processes, we have checked retention time and peak area replicates but ion selected is not corrected (shown in figure). Whether the above processes is correct? How to achieve the correct time alignment by one peptide set form experiment run? Can we do anything in Skyline to select the correct peaks?

sp|P00352|AL1A1_HUMAN Retinal dehydrogenase 1 OS=Homo sapiens GN=ALDH1A1 PE=1 SV=2 K.IFINNEWHDSVSGK.K [22, 35] Correct Correct Error Error