Each digested protein sample is labeled with a unique TMT™ tag. Samples are fractionated, purified, and then subject to liquid chromotography. (Image courtesy of Thermo Fisher Scientific).
Fractions from basic reverse-phase high performance liquid chromatography (bRP-HPLC) are collected and non-sequentially pooled prior to performing MS, contributing to improved depth of coverage.
Multi-notch MS3 quantitation, available only on Orbitrap™ Fusion™ systems, offers unparalleled accuracy of TMT™ quantitation. Multi-notch MS3 avoids the pitfall of underestimated fold changes in protein expression caused by contaminant reporter ion interference that occurs in traditional MS2 methods (1, 2). This is accomplished by synchronous precursor selection (SPS) and coisolation/cofragmentation of multiple MS2 fragments, increasing precursor ion capture, signal intensity, dynamic range, and accuracy of analysis. Ultimately, this means more proteins can be identified and accurately quantified.