Untargeted LC-MS Biomarker Discovery

Samples were subjected to untargeted LC-MS measurements using a TripleTOF 5600+ system (Sciex, Framingham, MA, USA) and MarkerView software (version 1.2.1, Sciex) [24 (link)]. The peaks table consisted of one normalized peak area column per sample, as well as a mass value (m/z, mass-to-charge ratio) and retention time (min) column common to all samples. The nonzero peak areas were converted to common logarithms. LMIs with good discriminative ability (e.g., distinguishing MRI (+)/cytology (+) from MRI (−)/cytology (−) groups) were identified using the logarithmic peak area. The procedures for assessing individual LMIs were as follows. (1) For each LMI, a discrimination threshold value was determined (using increments of 0.01), wherein the sum of the sensitivity and specificity was highest (if adjacent threshold values had the same highest discrimination performance, the mean value was used). (2) Individual LMIs showing good discrimination (a summed sensitivity and specificity of 160% or higher) were set aside for subsequent analysis. The same procedures as above were applied to discover individual LMI candidates for distinguishing MRI (+)/cytology (−) or MRI (−)/cytology (+) from MRI (−)/cytology (−) groups, after replacing the MRI (+)/cytology (+) group with the MRI (+)/cytology (−) group or MRI (−)/cytology (+) group.