Qualitative/cursory identifications in the putative mass and elution time (PMT tag) peptide library were based on a minimum cross correlation (Xcorr) score of 2 for all peptides identified at least twice in all MS/MS experiments.
All accepted peptides had to be fully tryptic, had a ΔCn ≥ 0.08, and had the minimum cross correlation (Xcorr) value of 1.9 (+1 charge), 2.2 (+2 charge), and 3.75 (+3 charge).
The minimum cross correlation coefficients (Xcorr) of 1.7 and 1.9 for a + 1 proteolytic peptide by trypsin and PK, 2.2 and 2.5 for a + 2 proteolytic peptide by trypsin and PK, and 3.3 and 3.75 for a + 3 proteolytic peptide by trypsin and PK, respectively, were proposed to meet a peptide identification false discovery rate (FDR) of ~1% threshold.
It is shown that the proposed code provides an optimal code length with minimum cross-correlation value when compared to other codes.
Instead of calculating mean cross-correlation values, we identified the minimum cross-correlation values associated with the presence of spurious pulses observed on one or a few sensors.
By contrast, cross-correlation coefficients never exceeded 0.5 for sensor pairs in which one trace had a spurious pulse (Fig. 13b d), whereas in the case of sensor pairs nominally recording the same data, and lacking spurious pulses, minimum cross-correlations coefficients never dropped below 0.5.
We used PhosphoPIC [42] to select for minimum cross-correlation (Xcorr) settings high enough to reduce the target false positive rate to less than 5%.
Preliminary filtering of identified peptides was performed using a minimum cross-correlation cut-off (Xcorr) of either 1.9, 2.2, or 3.75 for 1+, 2+, or 3+ charge states, respectively, for fully tryptic (peptides that contained either an arginine or lysine at the site of cleavage), partially tryptic, and non-tryptic peptides.
High confidence peptide identifications were obtained by filtering the search results based on the following criteria: minimum cross-correlation score of 1.8 (+1 ion), 2.2 (+2) and 3.5 (+3); minimum delta Cn of 0.1 and maximum probability of a random match of 0.01.
The minimum cross-correlation factor filter was readjusted for each individual charge state separately to optimally meet the predetermined target FDR of 5% based on the number of random false positive matches from the reverse decoy database.
Matched peptide sequences had to pass the following thresholds: 1) the uniqueness scores of matches (ΔCn) were at least 0.1 and 2) minimum cross-correlation scores XCorr of 1.9, 2.2, and 3.75 for charge states +1, +2, and +3, respectively.
