RESUMO
Charge detection mass spectrometry (CDMS) provides a direct measure of the mass of individual ions through nondestructive, simultaneous measurements of the mass to charge ratio and the charge. To improve the accuracy of the charge measurement, ions are trapped and recirculated through the charge detector. By substantially extending the trapping time, the uncertainty in the charge determination has been reduced by a factor of two, from 1.3 elementary charges (e) to 0.65 e. The limit of detection (the smallest charge that can be reliably measured) has been reduced by about the same proportion, from 13 to 7 e. The more precise charge measurements enable a substantial improvement in the mass resolution, which is critical for applications of CDMS to mixtures of high mass ions.
RESUMO
The assembly of hundreds of identical proteins into an icosahedral virus capsid is a remarkable feat of molecular engineering. How this occurs is poorly understood. Key intermediates have been anticipated at the end of the assembly reaction, but it has not been possible to detect them. In this work we have used charge detection mass spectrometry to identify trapped intermediates from late in the assembly of the hepatitis B virus T = 4 capsid, a complex of 120 protein dimers. Prominent intermediates are found with 104/105, 110/111, and 117/118 dimers. Cryo-EM observations indicate the intermediates are incomplete capsids and, hence, on the assembly pathway. On the basis of their stability and kinetic accessibility we have proposed plausible structures. The prominent trapped intermediate with 104 dimers is attributed to an icosahedron missing two neighboring facets, the 111-dimer species is assigned to an icosahedron missing a single facet, and the intermediate with 117 dimers is assigned to a capsid missing a ring of three dimers in the center of a facet.
Assuntos
Capsídeo/química , Capsídeo/metabolismo , Vírus da Hepatite B/fisiologia , Espectrometria de Massas , Montagem de Vírus , Vírus da Hepatite B/metabolismo , Modelos Moleculares , Conformação ProteicaRESUMO
Charge detection mass spectrometry (CDMS) measurements have been performed for cytochrome c and alcohol dehydrogenase (ADH) monomer using a modified cone trap incorporating a cryogenically cooled JFET. Cooling the JFET increases its transconductance and lowers thermal noise, improving the signal to noise (S/N) ratio. Single ions with as few as 9 elementary charges (e) have been detected. According to simulations, the detection efficiency for ions with a charge of 13 e is 75%, and for charges above 13 e the detection efficiency rapidly approaches 95%. With the low limit of detection achieved here, adjacent charge states are easily resolved in the m/z spectrum, so the accuracy and precision of the image charge measurements can be directly evaluated by comparing the measured image charge to the charge deduced from the m/z spectrum. For ADH monomer ions with 32 to 43 charges, the root mean square deviation of the measured image charge is around 2.2 e. Ions were trapped for over 1500 cycles. The number of cycles detected appears to be limited mainly by collisions with the background gas.