In-
solution enzymatic and nonenzymatic digestion methods have been successfully implemented in matrix-assisted
laser desorption/ionization-mass spectrometry (MALDI-MS)-based virus identification, extending to typing/subtyping of deadly influenza viruses. However, these methods are inefficient in obtaining more precise information on
surface proteins of myxovirus particles, not only the
hemagglutinin and
neuraminidase of influenza virus but also the
hemagglutinin-
neuraminidase of Newcastle disease virus (NDV). Imbalances in
viral protein composition cause ion suppression of tryptic fragments from low-abundant target
proteins (surface proteins), adversely affecting reproducibility of mass spectra. Additionally, the coexistence of tryptic
peptides from several
proteins requires sophisticated statistical solutions for precise result interpretations. To circumvent these, we apply
detergent-based (gel-free) partitioning of whole viruses into soluble
surface proteins and insoluble virus materials, using differential centrifugation. MALDI-TOF or MALDI-TOF/TOF MS was applied to analyze tryptic
peptides from separated
viral proteins. In this study, we achieved type/subtype of
avian influenza virus (AIV) within 5 h, based on 4 major
proteins, by significantly reducing ion suppression and signal overlap from various
protein sources. Hence, our approach can both yield dependable results and allow Web-based search engines to be directly employed, obviating the need for additional statistical strategy. Additionally, we demonstrate the utility of the method using NDV.