Streamlined processing and data acquisition for sample multiplexing

Isobaric labeling facilitates proteome-wide expression profiling across multiple samples within a closed system. A plethora of strategies have been published concerning sample processing and subsequent mass spectrometric data acquisition for isobaric tag-based experiments, including the streamlined-TMT (SL-TMT) protocol as highlighted here. This talk underscores several important aspects of such strategies, illustrates a basic sample processing and data acquisition framework, and suggests where further optimization may be necessary. Together, higher order multiplexing (TMTpro-18plex), high-field asymmetric-waveform ion-mobility spectrometry (FAIMS), and real-time database searching (RTS) can expand the depth, breadth, and accuracy of proteome-wide expression profiling. In addition, a single-step, medium-scale, phosphorylation experiment (“mini-phos”) can be integrated seamlessly into the workflow. This single spin column-based enrichment of the pooled TMT-labeled sample can routinely quantify several thousand phosphorylation sites from a TMTpro18-plex experiment starting from ~50 µg of protein per condition with minimal toll on time and cost. This talk showcases the general workflow using a TMTpro dataset in which the samples were processed with the SL-TMT protocol and the data were collected using FAIMS and RTS on an Orbitrap Eclipse mass spectrometer.