Proteins were designated putative binding partners if they met the following criteria: (i) possessed a minimum peptide probability cut-off of 0.85, (ii) were represented by two or more unique peptides, and (iii) were present in the experimental pTZ1066 but not the control pSE100 dataset.
Quantification parameters included: reporter ion tolerance – 0.05 Da; minimum peptide length – 6 amino acids; minimum # of spectra – 2; minimum # of peptides – 2; minimum peptide probability – 0.05; minimum protein probability – 0.5 and data centroided.
Scaffold data analysis (Proteome Software, Version 2.2.03) was conducted using Bioworks search file results with a high stringency filter with a 99% minimum protein ID probability, a minimum number of two unique peptides for each protein identified, and with a minimum peptide ID probability of 95%.
Proteins not meeting the minimum peptide number and probability requirement described previously were further removed from the list, resulting in 4 candidate targets each consisting of a single semi-tryptic peptide fragment (Table 1).
Minimum peptide and protein probabilities were set at 80 and 99%, respectively, with a minimum of five unique peptide matches per protein.
Minimum peptide and protein identification probabilities of 80 and 99% were required respectively, and a minimum of at least 2 unique peptide assignments to each identified protein.
Parameters included the following: mass tolerance of 20 ppm, minimum peptide length of 6 amino acids, protein probability of 0.5, and peptide probability of 0.05.
The minima criteria for protein identification were: peptide probability <1E-04; peptide XCorr vs charge = 1.5 for charge +1; 2 for charge +2; 2.5 for charge +3; 3 for charge +3; FDR <1%.
A p values of < 0.05 was considered significant, lists of identified proteins were made under the criterions, peptide probability >95%, protein probability >99% and 2 minimum unique peptides, and then were merged into a master file where the primary accession numbers and entry names from UniProtKB were used.
A multiple-threshold filter was applied at the peptide level: Xcorr magnitude were up to 1.7, 2.2, 3.3 and 4.3 for peptides with one, two, three and four isotopic charges respectively; peptide probability lower than 0.05, ΔCn >0.1 with a minimum of two different peptides for an identified protein.
The false discovery rate (FDR) was set to < 1.0% for both peptide and protein identifications, the minimum peptide length was set to 6. Phosphorylation site localization was based on PTM scores that assign probabilities for each of the possible sites according to their site-determining ions.
