As the well-established 'bottom-up' mass spectrometry–based approach continues its success in high-throughput proteomics, an emerging approach known as 'top-down' is beginning to make headlines, especially for the analysis of post-translational modifications (PTMs).

Deciphering the histone code could be aided tremendously by high-throughput top-down mass spectrometry. Credit: Nature Genetics

PTMs often occur in different combinations on individual proteins, and understanding these combinations is crucial for understanding biological regulation, such as for 'cracking' the histone code. In a bottom-up experiment, the proteomic mixture is digested into short peptides before analysis, so information about the correlated relationships of different PTMs is lost. Compounding the problem is the fact that many PTMs are unstable under typical mass spectrometry conditions, and that the mass spectrometer does not detect every last peptide, so minor PTMs occurring on a small percentage of proteins are often not observed.

In the top-down approach, intact proteins are introduced into the mass spectrometer, so important information about combinatorial PTMs is retained. In recent years, highly efficient fragmentation methods have been developed (such as electron capture dissociation and electron transfer dissociation) that are particularly good at preserving labile PTMs. The mass range of top-down has been extended to proteins as large as 229 kDa (Science 314, 109–112; 2006), and increasingly larger numbers of intact proteins can be detected in a single analysis. Yet top-down is still mainly a technique for analyzing single purified proteins.

Currently, larger sample quantities are required and the analysis time is longer than for a bottom-up experiment, precluding high-throughput analyses. New methods are needed for efficient protein separation, and robust computational tools for assigning protein identities and PTMs from top-down data are also lacking.

So stay on the lookout for new methods driving the limits of top-down mass spectrometry. Perhaps one day this approach will be the method of choice for investigating the biological importance of combinatorial PTMs.