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. 2014 Oct 30:4:6803.
doi: 10.1038/srep06803.

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry applied to virus identification

Adriana Calderaro  1 , Maria-Cristina Arcangeletti  1 , Isabella Rodighiero  1 , Mirko Buttrini  1 , Chiara Gorrini  1 , Federica Motta  1 , Diego Germini  1 , Maria-Cristina Medici  1 , Carlo Chezzi  1 , Flora De Conto  1
Affiliations

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry applied to virus identification

Adriana Calderaro et al. Sci Rep. .

Abstract

Virus detection and/or identification traditionally rely on methods based on cell culture, electron microscopy and antigen or nucleic acid detection. These techniques are good, but often expensive and/or time-consuming; furthermore, they not always lead to virus identification at the species and/or type level. In this study, Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) was tested as an innovative tool to identify human polioviruses and to identify specific viral protein biomarkers in infected cells. The results revealed MALDI-TOF MS to be an effective and inexpensive tool for the identification of the three poliovirus serotypes. The method was firstly applied to Sabin reference strains, and then to isolates from different clinical samples, highlighting its value as a time-saving, sensitive and specific technique when compared to the gold standard neutralization assay and casting new light on its possible application to virus detection and/or identification.

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Figures

Figure 1
Figure 1. Representative MALDI-TOF mass spectra of purified poliovirus Sabin reference strains and ClinPro Tools statistical analysis.
(a,b) Spectra of human poliovirus type 1, 2, 3 purified particles, shown in the m/z range of (a) 2 to 20 KDa (low molecular weight) and (b) 17 to 90 KDa (high molecular weight). Molecular weight values of poliovirus significant peaks are indicated on peak tops. (c,d) Analysis of discriminating peaks within the three poliovirus serotypes by using ClinPro Tools software (“Poliovirus type 1”, blue; “Poliovirus type 2”, red; “Poliovirus type 3”, green): “2D Peak distribution view” with the details at low (c) and high (d) molecular weight of the two best separating peaks; “PCA-3D dot plots window” of the spectra showing in 3 separate clusters the spectra of the 3 different poliovirus serotypes in the low (c) and high (d) molecular weight range; “Spectra view” in the low (c) and high (d) molecular weight range of the average spectra of the replicates of each poliovirus serotype. The red rectangles highlight the three discriminating peaks revealed in the mass range 2–20 KDa (7,522; 7,579; 7,635 m/z), and the four discriminating peaks in the mass range 17–90 KDa (30,197; 33,257; 33,554; 38,601 m/z). The blue rectangles are not discriminating peaks according to the applied Supervised Neural Network (SNN) statistical method.
Figure 2
Figure 2. Representative MALDI-TOF mass spectra of Sabin poliovirus-infected and uninfected LLC-MK2 cells and comparative analysis by ClinPro Tools software.
The spectra of LLC-MK2 cells infected with human poliovirus types 1, 2, 3 (“Poliovirus type 1-infected cells”, “Poliovirus type 2-infected cells”, “Poliovirus type 3-infected cells”), with pointed average masses, are compared in the m/z range 2 to 20 KDa (a–c) and 17 to 90 KDa (e–g) with the spectra of LLC-MK2 uninfected cells (d,h). (i,j) Spectra view of the Average spectra profiles in the mass range 7,400–7,900 Da (i) and in the mass range 26,000 to 35,000 Da (j) of LLC-MK2 cells infected with the 3 different poliovirus serotypes with LLC-MK2 uninfected cells.
Figure 3
Figure 3. Representative MALDI-TOF mass spectra of LLC-MK2 cells infected with poliovirus clinical isolates and of uninfected cells, and ClinPro Tools comparative analysis between clinical isolates and Sabin poliovirus type 1, 2, and 3 reference strains.
The spectra of LLC-MK2 cells infected with poliovirus isolated from different human samples (“sample 1 infected cells”, “sample 2 infected cells”, “sample 3 infected cells”), identified by neutralization assay as poliovirus type 1, 2 and 3, respectively, are compared in the m/z range of 2 to 20 KDa (a–c) with the protein profile of LLC-MK2 uninfected cells (d). Molecular weight values of poliovirus clinical isolates and cellular peaks are indicated on peak tops. (e–g) Spectra view of the average spectra profiles in the mass range 7,400–7,900 Da of the three Sabin reference poliovirus strains (green, blue and yellow for poliovirus type 1, 2 and 3, respectively) compared with the poliovirus strains isolated from clinical samples (violet; poliovirus type 1, 2 and 3, in panels e, f and g, respectively) and with LLC-MK2 uninfected cells (red). Rectangles highlight the match between the VP4 protein peaks of clinical isolates and those of the corresponding Sabin reference strain.
Figure 4
Figure 4. Representative MALDI-TOF mass spectra of LLC-MK2 cells infected with the Picornaviridae family members human coxsackievirus B1 and human echovirus 9 and uninfected cells, and ClinPro Tools comparative analysis with the Sabin poliovirus type 1, 2, and 3 strains.
(a) Spectra of LLC-MK2 cells infected with human coxsackievirus B1 and human echovirus 9 (“Coxsackie B1-infected cells” and “Echovirus 9-infected cells”) compared with the protein profile of LLC-MK2 uninfected cells in the m/z range of 2 to 20 KDa. Molecular weight values are indicated on peak tops. (b,c) Spectra view of the average spectra of human coxsackievirus B1 (b) and human echovirus 9 (c) compared by ClinPro Tools in the mass range 7,400–7,900 Da with the average spectra of the 3 Sabin poliovirus strains (poliovirus type 1; poliovirus type 2; poliovirus type 3) and uninfected LLC-MK2 cells.
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