Monitorizing structural integrity of Rotavirus like Particles using Mass Spectrometry Catarina Franco1, Marcos Sousa2, Carina Silva2, , Thomas J.D. Jørgensen4, Paula Alves1,2 Ana Varela Coelho1,3 1. Instituto de Tecnologia Química e Biológica, Oeiras, Portugal; 2.Instituto de Biologia experimental e Tecnológica, Oeiras, Portugal; 3. Universidade de Évora, Évora, Portugal; 4.University of Southern Denmark, Odense, Denmark Introduction Rotavirus infection is the most common cause of severe diarrhoea among children. Worldwide, it is responsible for the death of over 600.000 children annually, primarily in the developing countries. Virus-like particles (VLPs) represent an attractive alternative vaccine as they mimic the overall structure of the virus particles without the requirement of containing infectious genetic material. The produced Rotavirus VLPs are composed of 3 concentric protein layers: an inner shell consisting of Viral Protein 2 (VP2), a middle shell formed by Viral Protein 6 (VP6), and the outer shell is comprised of Viral Protein 7 (VP7). A complete disaggregation of the VLP into their constitutive proteins (VP2, VP6 and VP7) is crucial to further processing of the samples (namely target specific quantification and monitorization of structural integrity both by mass spectrometry experiments). Several degradation buffers were tested and the impact on rotavirus like particles was monitorized both by electron microscopy and by mass spectrometry (MALDI-linear mode). The dynamical properties of these viral proteins in the different types of VLPs were also investigated using amide hydrogen (1H/2H) exchange monitored by mass spectrometry. Material and Methods Viral capside of Rotavirus VP7 VP2 VP6 VP4 Virus-like particles References • ROMAN TUMA, LORI U. COWARD, MARION C. KIRK, STEPHEN BARNES AND PETER E. PREVELIGE JR, 2001, 306, 389-396, J. Mol. Biol., Hydrogen-deuterium Exchange as a Probe of Folding and Assembly in Viral Capsids. Particle degradation Moniturizing strutural integrity 1H/2H exchange in VLP 2/6/7 Method optimization VLP degradation Deuteration of VLP2/6/7 VLP 2/6/7 VLP 2/6 VP7 VP6 VP2 H2 H1 Sample treatment with 8M Guanidine Hydrochloride Sample treatment with 8M Urea 5uL of VLP2/6/7 + 95 uL D2O PBS (10 min) Sample desalting and concentration After degradation with the diferent reagents, sample was loaded in a GeLoader Tip containing either POROS R1 or C8 plug and eluted with Sinapinic Acid (20mg/mL) in 70% ACN 0,1% TFA directly to the MALDI plate Quenching the deuteration reaction Formic acid was added to reaction mixture until pH 2,2 Samples were then frozen in liquid nitrogen Determination intact mass m/z % % Degradation of VLP2/6/7 structure 8uL of Ureia 8M was added m/z Determination of incorporated H2 Linear-MALDI analysis Linear-MALDI analysis (After sample desalting) Results Method optimization Disagregation of VLP into their constitutive proteins 1H/2H exchange in VLP 2/6/7 Total incorporation of deuterium in VP7 and VP6 Control VP6 dVP6 BSA as a internal mass standard Degradation of the VLP´s with GuHCl Degradation of the VLP´s with Urea 30000 Intensit y 20 0000 30 min exposure Control <66431> <45040> <32885> <44853> <32589> dVP7 * dVP 6 BSA internal standard VP7 VP Mass spectra of VLP 2/6/7 before and after incorporation of deuterium. Highlighted in red (up) is the mass spectra of VLP 2/6/7 incubated in PBS with H2O as a control. In black (down) is the mass spectra of VLP 2/6/7 incubated for 10 min in PBS with D2O. It is possible to see clearly there is a mass increase in VP7 and VP6. For more detail see next picture. (* d stands for deuterated). MALDI-TOF mass spectra after treatment of VLP 2/6/7 with GuHCl and desalting with geloader tip packed with POROS R1 resin (20um). MALDI-TOF mass spectra after treatment of VLP 2/6/7 with Urea 8M and desalting with geloader tip packed C8 plug. Deuterium incorporation on VP7 m/z 31000 35000 200000 296 Da <32589> VP7 <32885> dVP7 * Control 30 min exposure Deuterium incorporation on VP6 Internal calibration of the spectra: Trying to obtain better mass accuracy Deuterium incorporation in VP6. Highlighted in red is the mass spectra of VP6 in VLP2/6/7 incubated in PBS with H2O as a control. In black is the mass spectra of VP6 in VLP 2/6/7 incubated for 10 min in PBS with D2O. It is possible to see clearly there is a mass increase of 186 Da in VP6. m/z 43000 47000 90000 <45040> dVP6 <4485 4 > VP6 18 6 Da Control 30 min exposure MALDI-TOF mass spectra of VLP 2/6/7 after performing internal calibration. BSA as used as an internal standard (average mass 66431 Da). The mass of VP6 in the spectra has a difference of 6 Da from the predicted mass (44842 Da, VP6 form RF strain). Deuterium incorporation in VP7. Highlighted in red is the mass spectra of VP7 in VLP 2/6/7 incubated in PBS with H2O as a control. In black is the mass spectra of VP7 in VLP 2/6/7 incubated for 10 min in PBS with D2O. It is possible to see clearly there is a mass increase of 296 Da in VP7. Conclusions 1H/2H exchange in VLP 2/6/7 •We can see perfectly VP7 and VP6 and just a very little signal from VP2 in MALDI in the treatment of VLP 2/6/7 with GuHCl and urea. The intact masse of VP6 was a deviation of 100 Da from the mass predicted directly from the sequence (44842 Da). This suggests that the external calibration is not sufficient for mass accuracy, so an internal calibration is preferable. •In order to obtain better mass accuracy for the viral proteins an internal calibration was preformed. For this purpose a standard protein with a known exact mass (BSA, 66431 Da average mass, MALDI mass standard, PerSeptive Biosystems) was added to the MALDI matrix so that in the spectrum should appear both masses of the viral proteins and the standard protein. •Mesurements higly dependent on the internal calibration (if for any reason the signal of the internal standard is poor, accuracy decreases); •The method is also compatible with the viral proteins VP7 and VP6 but we cannot see VP2 due to the deficient matrix crystallization associated with this procedure (rapid crystallization in vacuum generates poor co-crystallization between matrix and analyt). Future work: Differences in deuterium incorporation on VP6 in both VLP2/6/7 and VLP2/6 will be compared in order to determine if this is a valid method for determination of stability of VLP 2/6/7. Acknowledgments This work was financed by Fundação para a Ciencia e Tecnologia Project POCI/BIO/55975/2004