These methods were optimized as previously

described with

These methods were optimized as previously

described with some modification [27]. For both methods, each mass spectrum was obtained from the sum of 10 scans of 150 laser shots each and using 512 K data points. Typically, the target plate offset was 100 V with the deflector plate set at 180 V. The ion funnels operated at 100 V and 6.0 V, respectively, with the skimmers at 15 and 5 V. The analyzer entrance was maintained at −7 V, and side kick technology was used to further optimize peak shape and signal intensity. The two acquisition settings differentiate for the trapping potentials (LM, 0.6 and 0.55 V; this website HM, 0.95 and 0.80 V), the required excitation power (LM, 25%; HM, 28%) and pulse time (LM, 10 μs; HM, 20 μs), the time of flight to the ICR cell (LM, 1.350 ms; HM, 2.700 ms) and the quadrupole filter mass (LM, m/z 1300; HM, m/z 2500). For each spotted sample, two duplicate spots were measured using the LM and the other two using the HM. Approximately 4.5 h were needed to measure 384 MALDI spots (i.e. originating from 96 different serum samples). DataAnalysis Software 4.0 SP 5 CH5424802 price (Bruker Daltonics) was used for the visualization and the calibration of the spectra. Prior to the measurement of each MALDI plate the FTICR system was externally

calibrated using a commercially available peptide mix and a protein mix (Bruker Daltonics). The spectra obtained using the LM were internally calibrated only when used for identification purposes. The m/z-values used for the internal calibration of the LM and the HM are reported in Table S1 in the Supplementary Material. Peaks were

determined using the FTMS algorithm with a signal-to-noise threshold of 3 and using the centroid for peak position with a percentage height of 80. Protein and/or peptide signals in RPC18 profiles were quantified as follows. First, based on visual inspection of the profiles, 457 and 670 peaks were selected for the LM and HM spectra, respectively, for further analysis. To this end, a so-called reference file was compiled for both types of profiles in such a way that for each selected peak the m/z-value, Vasopressin Receptor a peak number and an m/z-window were reported. In the LM profiles, this m/z-window ranged from 0.015 to 0.166 Da while in the HM it ranged from 0.05 to 0.31 Da reflecting the peak width along the spectra. Then, the in-house developed Xtractor tool was used to determine the intensity of each user-defined peak. This open source tool generates uniform data (peak) arrays regardless of spectral content ( MALDI-FTICR profiles were exported as XY (.xy) files, all containing m/z values with corresponding intensities. Although peptide and proteins were measured up to 10,000 Da using the HM method, the peak selection was limited to 9043.3 Da. The analysis of the spectra in the m/z-range from 9043.3 to 10,000 is on-going and the results will be presented in a separate study.

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