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 (TM)) 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. (C) 2010 Elsevier B.V. All rights reserved.