Top-down analysis of small plasma proteins using an LTQ-Orbitrap. Potential for mass spectrometry-based clinical assays for transthyretin and hemoglobin

Transthyretin (TTR) amyloidosis and hemoglobinopathies are the archetypes of molecular diseases where point mutation characterization is diagnostically critical. We have developed a top-down analytical platform for variant and/or modified protein sequencing and are examining the feasibility of using this platform for the analysis of hemoglobin/TTR patient samples and evaluating the potential clinical applications. The platform is based on a commercial high resolution hybrid orbitrap mass spectrometer (LTQ-Orbitrap™) with automated sample introduction; automated data analysis is performed by our own software algorithm (BUPID top-down).The analytical strategy consists of iterative data capture, first recording a mass profile of the protein(s). The presence of a variant is revealed by a mass shift consistent with the amino acid substitution. Nozzle-skimmer dissociation (NSD) of the protein(s) yields a wide variety of sequence-defining fragment ions. The fragment ion containing the amino acid substitution or modification can be identified by searching for a peak exhibiting the mass shift observed in the protein mass profile. This fragment ion can then be selected for MS/MS analysis in the ion trap to yield sequence information permitting the identification of the variant. Substantial sequence coverage has been obtained in this manner. This strategy allows for a stepwise MS/MS analysis of the protein structure. The sequence information obtained can be supplemented with whole protein NSD fragmentation and MS/MS analysis of specific protein charge states. The analyses of variant forms of TTR and hemoglobin are presented to illustrate the potential of the method.

Graphical abstractFigure optionsDownload full-size imageDownload high-quality image (154 K)Download as PowerPoint slideResearch highlights▶ Top-down sequencing of proteins for detection of variants in plasma proteins. ▶ Post-translational modifications also defined and located. ▶ Nozzle-skimmer dissociation followed by CID. ▶ Approach should have potential for clinical use.