|R. Théberge,W. Tong, G. Infusini, Mark E. McComb, Catherine E. Costello.|
Center for Biomedical Mass Spectrometry, Department of Biochemistry, Boston University School of Medicine, Boston MA.
|Introduction: Hemoglobinopathies and transthyretin amyloidosis are the archetype of molecular diseases where the characterization of point mutations is diagnostically critical(1). We have developed a top-down analytical platform and are examining the feasibility of using this platform for analysis of hemoglobin/TTR patient samples as well as evaluating potential clinical applications.|
Method: Hemoglobin samples are obtained by diluting whole blood. Transthyretin is isolated by immunoprecipitation from patient serum The samples are introduced into the Thermo-Fisher LTQ-Orbitrap hybrid MS using an Advion Triversa NanoMate ESI source. Fragment ion mass spectra are generated by nozzle-skimmer dissociation (NSD), LTQ-CID and C-trap dissociation. MS/MS spectra are analyzed using BUPID-Topdown (Boston University Protein Identifier-Topdown), a custom-programmed software algorithm written in-house (2).
Results: The analytical strategy begins with recording a mass profile of the protein. The presence of a variant or post-translational modification (PTM) is revealed by a mass shift consistent with the amino acid substitution or modification. Nozzle-skimmer dissociation (NSD) of the protein 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 mass selected for MS/MS analysis in the LTQ to yield sequence information that permits identification of the variant or PTM. NSD voltage can be modulated to favor the production of specific fragment ions, e.g., NSD of TTR yields the sequence-defining complementary ion pair y85 9+ and b42 6+ and these primary fragments have been used as precursors for subsequent MS/MS analysis. 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. A similar approach is being used for hemoglobins, although the presence of two components (alpha and beta chains) complicates matters. The synergy of instrument, sample introduction system and data analysis software offer streamlined capabilities for the analysis of small proteins in order to characterize sequence variants and PTMs.
1. C. R. Scriver et al, eds. The Metabolic and Molecular Bases of Inherited Disease (.., 8th ed., McGraw-Hill, New-York, 2001.
2. W. Tong et al., BUPID-Top-Down: Database Search and Assignment of Top-Down MS/MS Data. 57th ASMS Conference, Philadelphia , June 2009.
This research is funded by NIH-NCRR P41-RR10888, S10-RR20946 and NIH-NHLBI N01-HV28178.