Flow-through of Current Phospho-proteomic Analysis. Using phosphoenrichments (e.g. IMAC, TiO2; SIMAC) we are capable of isolating phosphorylated peptides and discard un-phosphopeptides. The isolated phosphopeptides have to be cleaned and desalted via chromatography (e.g. POROS R3, Disks C18 or graphite-, which isolate hydrophilic peptides) before the MS analysis. Finally, the desalted and cleaned phosphopeptides are injected into the mass spectrometer. Different types of ionization can be used (e.g. Matrix-Assisted Laser Desorption/Ionization MALDI or ElectroSpray Ionization ESI). Also, different kinds of fragmentations can be used (e.g. CID, ETD, ECD). In addition, different MS modes can be useful, for example: MS/MS, MSA, MS3NL. As a general rule, positive MS mode is currently more efficient than negative mode for phosphoproteomic studies. It is always necessary to test and combine different phosphoenrichments together with different MS strategies to recover and identify the maximum level of phosphopeptides. This will imply a high efficiency for your clinical research study. The resulting data (phosphorylated proteins identified) must be coupled to bioinformatic tools (software) in order to improve the biological understanding.