Sorting Out Ubiquitination
“We are following the process of polyubiquitination through the use of mass spec,” stated Eric Dammer, Ph.D., a postdoctoral fellow in the lab of Junmin Peng, Ph.D., assistant professor in the department of human genetics at Emory University School of Medicine, “with the long-range goal of developing new drugs for treating mental disorders.”
In order to investigate the ubiquitin trafficking process, Dr. Dammer and his colleagues used mass spec combined with SILAC (stable isotope labeling with amino acids in cell culture). This is an ideal technology for establishing linkages of polyubiquitin to target proteins, but it requires careful optimization of the LC/MS/MS parameters, including loading amount and gradient length.
By systematically adjusting these properties, they were able to identify proteins and investigate relationships between loading amounts and gradient lengths that can maximize protein identification. Knowing the appropriate settings and parameters is critical in mass spectrometry as frequently sample amounts are limited and precious.
The group did these investigations using the Thermo Scientific LTQ Orbitrap XL ETD (Thermo Fisher Scientific), which was specifically designed for proteomics analyses. It combines three different and complementary fragmentation techniques, collision-induced dissociation, higher-energy collisional dissociation, and electron transfer dissociation.
Sample preparation for mass spec analysis is a critical component of the technology, since the full potential of the hardware cannot be realized without high-quality preps. Moreover, in today’s laboratories high throughput is also essential in order to match the rapid analysis of which the instruments are capable. For this reason these developments will be followed with great interest in the coming years by those in the field.