In total, 273 serum samples were analyzed in this way, thus yielding 1092 profiles. A typical example of both an LM and HM MALDI-FTICR profile is depicted in Fig. 1A. It was verified

that all peptides and (small) proteins were measured with isotopic resolution through all the spectra, with typical resolving powers varying from 130,000 (m/z 1039.6727) to 46,000 (m/z 3523.7664) in the LM spectra and from 150,000 (m/z 3680.8709) to 33,000 (m/z 9744.6054) in the HM spectra (as plotted in Fig. 2A). As a result, a large number of peptides or proteins that would overlap in high resolution MALDI-TOF MS were measured as distinct features by MALDI-FTICR MS. Two examples of resolved species are shown in Fig. 2B, selleck chemical one for the LM and one for the HM profiles. The ultrahigh resolving power allowed the accurate quantification of the selected peptides and proteins in all the spectra. After manual inspection of the profiles, 457 and 670 peaks for the LM and HM, selleck compound respectively, were selected for statistical analysis. After taking into account isotopic peaks from the same species, 196 peptides remained from the 457 selected peaks in LM spectra and 291 peptides or proteins remained from the 670 selected peaks in HM spectra. Peptides and proteins were detected with signal intensities that typically

ranged over two orders of magnitude. For example, Fibrinopeptide alpha chain (2–16) (at m/z-value 1465.6554) was often observed as the most intense peptide, and was 304 times more intense than Complement C4-A (1337–1350) (at m/z-value 1626.8459) detected with a signal-to-noise ratio (S/N) of 6.6, in a typical spectrum. Thus, peptides observed with low S/N were also evaluated. Interleukin-3 receptor For example, the peptide identified

as oxidized Fibrinogen beta chain (45–71) (m/z 2898.5334) (see Section 3.3) was observed in the spectra in the calibration set with an averaged S/N 9.6 with a standard deviation (SD) of 6.4, while the highly intense Complement C3f fragment peptide (at m/z-value 2021.1039) was observed with an averaged S/N of 2035 with an SD of 345. As a final remark, from 12 out of 1032 profiles the quality was insufficient for further statistical analysis, most likely because of failed MALDI spotting. The signal intensities of all selected peaks were determined in all serum profiles using the Xtractor tool described in the Materials and methods section. As shown in Fig. 2A, the m/z-windows in the reference files were fine-tuned according to the resolving power calculated for each m/z-value. The presence of different peptides with close masses was also taken into account as well as the mass measurement precision (see Fig. 2B). The optimization of this m/z-window allowed the accurate quantification of all peaks selected from the spectra. Thus obtained peak intensity values were then used for statistical analysis.