Targeted Analysis
Targeted profiling is based on high mass accuracy measurements (~2 parts per million) using either an Q-Exactive Plus or an Ascend Multiomics Orbitrap mass spectrometer, polarity-switch and retention times (RT), the latter established by analyzing standards available in the Center.
‘Super-targeted’ analysis is also available when targeting a limited set of molecules where increased sensitivity and specificity is needed. For such an analysis Selected Ion Monitoring (SIM), Parallel reaction monitoring (PRM) or both is used.
Ion traces are typically extracted using SkyLine, a open-source, user friendly and freely available software developed by the MacCoss Lab at Washington University.
Targeted analysis data extracted using Skyline. From Left to right: molecules in our library, Extracted MS1 signal in positive and negative mode with Explicit retention time marked, MS1 signal for the measured molecule in different samples, Measured retention times of the molecule different samples.
Targeted analysis is often referred to as a Level 1 identification.
Level 1 through 5 type identification of metabolite identifications levels as described by Schrimpe-Rutledge et al: Level 5: Unique feature, Feature can be compared between samples. Level 4: Features are assigned possible atomic composition based on accurate mono isotopic mass, isotope distribution and charge. Level 3: Tentative structure is assigned based on additional information, for example Retention Time. Level 2: Putative identification based on fragmentation, for example with match to library database. Level 1: Validated identification based on standard which can be heavy isotope labelled.
Available standards includes: