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Processing software can detect the problem by comparing the measured distribution with the theoretical one for the most intense data in the data set.
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Of the four highly-specific antibodies we produced, Mab 24 2.3 was also selected for further immunohistochemical experiments because it showed the cleanest and most intense staining (data not shown).
Peptides were eluted with a linear gradient of buffer B; 4% B was first applied, followed by a linear gradient to 50% B in 105 min, and then to 100% B in 9 min, holding at 100% B for an additional 6 min. MS/MS spectra were acquired using a full scan followed by five MS/MS scans on the five most intense precursor ions in data-dependent mode.
Building these methods requires a library of MS/MS spectra for inclusion of the most intense transition pairs (m/ z data for parent ion and sequence fragment ion) for a given peptide.
The four most intense ions were selected in data-dependent mode for fragmentation in the linear ion trap, for details see [ 47].
The four most intense ions were selected in data-dependent mode and fragmented in the linear ion trap, with settings as in Ali, et al. [ 58].
For PMF data, the 100 most intense peaks were submitted to Spectrum Mill using a search tolerance of 25 ppm.
For every sample and preselected calibrant, the top 3 signals from 4 m/z window around the calibrant and most intense scan were picked as matching measured data.
BLS from the region of interest (ROI) was expressed using the pseudo colour scale (Red most intense and Blue least intense luminescence) and the data were presented as the cumulative photon counts collected within each ROI.
The acquisition of data was performed on a MassLynx data system (version 4.0; Waters) using a data-dependent mode where the four most intense precursors in a survey scan were isolated for collision-induced dissociation.
High resolution, accurate mass data were acquired in a data-dependent manner, with the top 20 most intense peptides in each MS scan selected for fragmentation.
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