One of the challenges in using mass spectrometry for proteomic research is distinguishing between molecules that are structurally different, but have the same mass.
In a recently published paper, researchers at the Neuroproteomics Laboratory at the University of Campinas (UNICAMP) report that they have created an optimized method that increases the resolution of proteomic analysis by mass spectrometry. The technique, which combines two-dimensional chromatography and ion mobility, was used to identify 10,390 proteins expressed in oligodendrocytes, the central nervous system cells that produce myelin. A previous study done by the group using single-dimensional chromatography for pre-sorting only yielded 2,290 proteins. The team is interested in oligodendrocytes for their possible role in schizophrenia.
The first step in the process is to break the oligodendrocytes into peptides. Next chromatography is used to separate the sample. For this experiment, two matrices were used instead of the typical one matrix separation. In the first separation, the peptides in liquid form are injected into the spectrometer in batches, with one fifth of the sample going in at a time.
Once inside the spectrometer, the sample is transformed into gas and flies back and forth in a vacuum, smaller peptides reach their destination faster and their mass is measured first.
"The resistance offered to the gas by the molecule depends on its three-dimensional shape, so if two different peptides with the same mass are flying together and we inject the gas in the opposite direction, they will tend to be separated by the force of resistance to the gas. It's like picking up two sheets of paper with the same mass, crumpling one into a ball, and dropping them both. Because of its shape, the crumpled sheet will reach the floor first," explained Martins-de-Souza who heads the UNICAMP laboratory.
The experiment yielded 223,000 peptides, which were reconstructed using bioinformatics tools, to create the 10,390 proteins described. Bioinformatics was also used to map the cellular compartments where the proteins are located and the biological processes they are involved in. With this technique, even relatively scarce proteins were able to be identified.