Middle-down sMRM HR acquisiton method setup using EAD fragmentation on the ZenoTOF® 7600 system

For research use only. Not for use in diagnostic procedures.

Answer

The top-down and bottom-up proteomics workflows are well established for characterizing either intact proteins or peptide fragments after proteolytic digestion, respectively. However, the middle-down workflow for electron-activated dissociation (EAD)-fragmentation on a ZenoTOF® 7600 system is a new approach that efficiently characterizes relatively large protein fragments and IgG subunits by obtaining higher sequence coverage than traditional approaches that relied on multiple fragmentation techniques. EAD MS/MS experiments produce extensive, information-rich fragments that provide better spectral coverage for larger peptide and subunit sequences than collision-induced dissociation (CID) MS/MS. When coupled with the advanced algorithms found in Biologics Explorer software that can analyze, interpret, and annotate the more complex MS/MS EAD data, complete sequencing of larger peptides and subunits can be obtained in a single injection. 

An overview of the EAD-based middle-down workflow from sample preparation to data acquisition and analysis is shown below.

In the EAD-based middle-down workflow (pictured above), an intact NIST mAb was proteolytically digested into the F(ab)’ and Fc/2 subunits; subsequent reduction produced the subunits Fc/2, LC and Fd subunits, which were chromatographically separated and then fragmented by EAD in a ZenoTOF 7600 system. Biologics Explorer software was used to interpret the EAD MS/MS spectra of the mAb subunits, producing an annotated map of the sequence.

The picture below shows the chromatograms of the subunit proteins (Fc/2, LC, and Fd) and the MRM HR-specific charge states of the protein envelope. Choosing a low, medium, and high charge state of the protein envelope is critical to get the most diverse fragmentation. The example below uses three different charge states to create an sMRM HR method.

A typical sMRM HR acquisition method with EAD fragmentation is shown below for the chosen charge states (above). The red circled parameters are specific for a middle-down high resolution MRM acquisition method that employs EAD fragmentation and are a good starting point for a successful middle-down approach.

Finally, the data files for each subunit can be processed in Biologics Explorer. All results from the middle-down analysis are loaded into the easy-to-use, optimized review template after processing in the workflow template. By reviewing the results in one template, they can be compared using a sequence coverage map, summary table and a combined MS/MS spectrum. The review template also facilitates sequence confirmation or PTM locations through the comparison of MS/MS spectra by overlaying or stacking the spectra using mirror plots.