18MDa intact virus capsid is the heaviest object to be analysed so far

It's not quite the ‘elephant in the room’, but an 18 megadalton viral assembly is perhaps the biggest thing in the mass spectrometer (MS). Dutch and US researchers have used quadrupole time-of-flight (QToF) native MS to investigate intact capsids from a bacteriophage – a virus that infects bacteria. While there is theoretically no upper limit on the mass of a particle that might be analysed using ToF MS, the work is far from trivial in breaking through the record.

The late John Fenn shared the 2002 Nobel prize in chemistry for his pioneering work on electrospray ionisation techniques in mass spectrometry, which paved the way for it to handle larger and larger molecules. Critically, the techniques that have emerged during the last decade or so allow protein complexes and even viral particles to be carried unharmed into the gas phase where they can then be subjected to analysis. Native MS has recently been exploited in just such studies looking at intact viruses and viral capsids, which are important in the spread of viral disease but also as structures that might be exploited in nanotechnology and drug delivery.


The record-breaking virus capsid is made up of 420 40kDa sub-units, giving it an impressive molecular formula

Albert Heck of Utrecht University in the Netherlands and colleagues there and at the Scripps Research Institute, La Jolla, California, have applied this analytical approach to bacteriophage HK97. This is an important model for understanding viruses that infect bacteria. They explain that the capsid assembles in vitro from a mixture of pentameric and hexameric capsomers made from protein gp5 and viral protease gp4. This builds an icosahedral intermediate, Prohead-1, which in nature matures by swelling up through activation of the protease molecules. However, the researchers could trap the growing capsid at the Prohead-1 stage for the investigation simply by leaving the proteases out of the recipe and over-expressing the gp5.

The results they have now obtained represent the biggest viral entity yet studied by mass spectrometry. The mass of the monomer building blocks are revealed to be above 40 kilodaltons, while the pentameric and hexameric capsomer assemblies are more than 210 kilodaltons and almost 253 kilodaltons, respectively, the team reports.

‘Given that the capsid comprises 420 gp5 units, we estimate the record-breaking mass to be close to 18 megadaltons. Indeed, we measured an experimentally accurate mass of 17.942+/-0.004 megadaltons,’ Heck tells Chemistry World.

The researchers suggest that there are various tweaks, such as improved desolvation, that might be made to a QToF instrument to further improve resolution allowing other protein complexes and viral particles as large as 20 MDa to be studied with good resolution.

Angela Corcelli, of the University of Bari Aldo Moro in Italy, is involved in complementary work that focuses on the lipid components of intact viruses. ‘Mass spectrometry of intact biological samples such as isolated membrane domains, organelles and viruses, represents an innovative analytical approach, which will provide novel extremely valuable information and data on biological structures,’ she says.

‘Heck's technique opens new exciting developments – not only in the study of proteins, but also in that of lipid-protein complexes and lipids of biomembranes.’