Typical parameters employed for MIDA were as follows: mass tolerance of ±10 ppm; minimum S/N of 100; [M + H]+ or [M – t-butyl]+ target ion for methane PCI and EI, respectively; 1 target per MS spectrum; and no dynamic exclusion of targets (0 s exclusion).
where x ̃ t - 1 k stands for the estimation of the PF at time t - 1 for target k and ϕ is the blocking function defining exclusion zones that penalize particles from target l falling into the exclusion zone of target k.
LC-MS/MS data were acquired in a data-dependent fashion by selecting the 4 most intense peaks with charge state of 2 to 4 that exceeds 20 counts, with exclusion of former target ions set to "360 seconds" and the mass tolerance for exclusion set to 100 ppm.
An Information Dependant Acquisition (IDA) experiment was used to specify the criteria for selecting each parent ion for fragmentation which included selection of ions in m/z range: >400 and <1600, of charge state of +2 to +5, exclusion of former target ions for 30 seconds, accumulation time of 1 second for a full scan and 2 seconds for MS/MS, ion source voltage of 2200 V and temperature 120°C.
The acquisition cycle consisted of a high-resolution precursor ion spectrum over the m/z range 350 1500, followed by up to 20 CID spectra (with a 60 s dynamic exclusion of former target ions).
SERRATE encodes a zinc finger protein required for miRNA biogenesis and RNA splicing while AMP1 associates with ARGONAUTE1 at the endoplasmic reticulum and is required for translation inhibition through the exclusion of miRNA target mRNAs from polysomes [ 19, 20, 23].
Since this tool is known to also have unspecific effects, control experiments are needed, chiefly among them the exclusion of off-target effects and the reconstitution of the genes' expression for the rescue of the cellular RNAi effects.
An additional condition is the exclusion of irrelevant target-probe pairs with >18 consecutive nucleotide matches.
A likely explanation for exclusion of these targets by OliCheck is the occurrence of highly homologous (>98% nucleotide identity) sequence repeats found in other ORFs.
Interestingly, 23 of the 25 compounds with a predicted pIC50 value higher than 7 were already predicted to interact with DHFR by the target prediction algorithm (Figure 4) at the exclusion of any other target.
