Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (FE SW 11.1) using one color scan setting for 1x44k array slides (Extended Dynamic Range, Scan Area 61×21.6 mm, Scan resolution 5 μm, Dye channel is set to Green and Green PMT is set to XDR Hi 100%% and XDR Lo 10%%).
CNT array treatment was achieved by scanning line-by-line at 512 lines per scan resolution.
The scan was collected under absorbance mode from 4000 to 400 cm−1 at 4 cm−1 scan resolution.
Step Size (ps) is the waveform scan resolution (step size between points), in picoseconds (1 bin = 3.18 pS).
The top ten most intense precursor ions from each full scan (resolution 70,000) were isolated for MS2 analysis.
The resulting grossly different scan resolution value caused the candidate patch filtering process to fail and further prevented the correct recognition of the color reference targets.
The top 15 most intense precursor ions from each full scan (resolution = 70,000) were isolated for HCD MS2 (resolution = 17,500; normalized collisional energy = 27) with a dynamic exclusion time of 20 s.
For each measurement set, scans were acquired for duration of around 1 min. The number of scans acquired depends on the scan rate which in turn depends on the waveform scan resolution, the window size, and the Integration size.
Top 20 precursor ions were selected into the HCD chamber for MS2 fragmentation analysis, MS/MS scan resolution was 17,500, AGC was set as 5e 4, the maximum injection time was 80 ms.
Bone metastasis was also confirmed on the final day by high-resolution microCT on a SCANCO vivaCT 75 microCT with 20.5 μm scan resolution (Scanco USA, Inc., Wayne, PA).
For scan resolution, pixel size was set as 50 µm.
