Identification of viral mutants by mass spectrometry

Abstract

A method to identify mutations of virus proteins by using protein mass mapping is described. Comparative mass mapping was applied to a structural protein of the human rhinovirus Cys1199 --> Tyr mutant and to genetically engineered mutants of tobacco mosaic virus. The information generated from this approach can rapidly identify the peptide or protein containing the mutation and, in cases when nucleic acid sequencing is required, significantly narrows the region of the genome that must be sequenced. High-resolution matrix-assisted laser desorption/ionization (MALDI) mass spectrometry and tandem mass spectrometry were used to identify amino acid substitutions. This method provides valuable information for those analyzing viral variants and, in some cases, offers a rapid and accurate alternative to nucleotide sequencing.

Figure 1

Schematic representation of the method to identify virus mutants by mass spectrometry. (Top) The observation of differences between wild-type and mutant viruses indicates amino acid changes (i.e., mutations). (Middle) Proteolysis of the viral variants generates a set of digestion fragments (one or more of which may contain the mutated amino acid), which appear at different m/z values as a consequence of an amino acid change(s). This observation provides valuable information as to the location of the mutation, augmenting its identification either by further mass spectral analysis (Bottom spectra) or nucleotide sequencing. (Bottom) Tandem mass spectrometric experiments (on the fragment containing the mutation) generate sequence information providing definitive identification of the mutated amino acid.

Figure 2

Comparison of the MALDI-TOF mass spectra resulting from the trypsin digestion of wild-type TMV and its mutant TMV-Asp50Arg. Most ion signals are common to both spectra, with the obvious exception of the signals observed at m/z 2050.5 Da in the wild-type spectrum and m/z 2091.8 Da in the mutant’s spectrum. Both signals represent residues 72–90 with the incorporation of the Asp → Arg amino acid change in the mutant. The mutation produces an ion signal (m/z 2091.8) 41 Da higher in mass than the corresponding signal from the wild-type virus. (Inset) Spectra from the trypsin digests of wild-type HRV14 and its naturally occurring mutant HRV14-Cys1199Tyr. Similarly with the HRV14 spectra, most fragments are common to both spectra, with the exception of the ion signal at m/z 4700.5 Da and 4783.8 Da in the HRV14 and HRV14-Cys1199Tyr spectra, respectively. Both ion signals corresponds to residues 187–227 of the VP1 capsid protein, and the mass difference of 83 Da between the two variants is consistent with a Cys → Tyr mutation. Mass analyses of the digestion mixtures resulted in the localization of the mutations in only a matter of minutes (<20 min, including the digestion).

Figure 3

High-resolution MALDI FTMS mass spectra of wild-type TMV and TMV mutants. (Upper) Spectrum represents the trypsin digest of wild-type TMV-U1. The mass accuracy of these fragments was within 2 ppm with an external calibrant. (Lower) Comparison of the digests of mutants TMV-Glu50Gln (Left) and TMV-Asp77Asn (Right) with wild type reveals mass differences of 1 Da from wild type in both cases. These mass differences are accordant with the corresponding mutations (as their names indicate). The high-resolution mass measurement allowed for the localization of the mutated amino acids.

Figure 4

Tandem mass spectral analysis of trypsin fragment 47–61 (m/z 1769.9361 Da) of mutant Glu50Gln. Both C-terminal (m/z 1641.86 Da) and N-terminal fragmentation (m/z 1296.76 and 1478.80 Da) confirm the identity of the mutation as Glu50Gln.