Both the top-down and bottom-up proteomic strategies have advantages and disadvantages, depending on the specific protein of interest. The bottom-up approach, for example, is easily used in high-throughput analysis; however, top-down methods allow for the analysis of intact proteins. Both methods have difficulties differentiating among protein isomers. An intermediate approach to these two methods is the middle-down approach to characterizing proteins. The middle-down approach is well suited to identify more complex proteins and proteins greater than 3,000 Da, such as histones.1
Advances in mass spectrometry (MS) have made it possible for the middle-down approach to also accurately characterize protein isoforms, variants and combinations of post-translational modifications. MS fragmenting methods such as electron capture dissociation (ECD), used alongside the middle-down approach, allow sequencing of highly modiﬁed and large polypeptides by increasing the coverage of peptide sequences and the retention of labile post-translational modifications.2
In their recent publication, Kalli et al. (2013) demonstrated the effectiveness of the middle-down approach in combination with ECD by characterizing histone proteins.1 Calf thymus, HeLa, Jurkat and MCF7 cells provided unfractionated histones for study. Proteins present were digested with Glu-C and Asp-N, which cleaved less frequently, allowing analysis of large peptides.3
Researchers employed a nanoﬂow liquid chromatography (nLC) system, an EASY-nLC II coupled to a 7T LTQ FT-ICR Ultra mass spectrometer equipped with a nanoelectrospray ion source (all from Thermo Scientiﬁc). The LTQ FT-ICR was able to alternate between a full scan (m/z range 300−1700) and ECD−MS/MS scans of the three most abundant ions using the data acquisition mode. After spectrometric raw data ﬁles were obtained, the files were deconvoluted with MS-Deconv. The researchers also utilized a database developed in-house for peptide and protein identiﬁcation,4 and compared the results of the MS analysis with a custom database derived from UniProt sequences for all human or bovine histones and contaminant sequences. Both the peptide and protein levels were validated, with a false discovery rate of less than 1%.
Using ECD in combination with a middle-down LC−MS/MS approach proved to be a highly efficient and effective method of characterizing histones. A single scan produced product ions with sufficient signal intensity and a high coverage of peptide sequences, and the majority of the identiﬁed peptides were in the mass range from 3 to 9 kDa.
A frequent challenge in identifying histones is the number of existing variants. For example, histones H2A 2-A and H2A.1 differ only in amino acids at the positions 16 and 51; yet the researchers easily differentiated and identified these histones, along with other peptides having single, multiple or isomeric post-translational modification sites. Based on the results of the middle-down LC−MS/MS approach developed by Kalli et al., this method appears highly effective for analyzing histones and other large and highly modified peptides.
1. Kalli, A., Sweredoski, M.J., and Hess. S. (2013) “Data-dependent middle-down nano-liquid chromatography-electron capture dissociation-tandem mass spectrometry: An application for the analysis of unfractionated histones,” Analytical Chemistry, 85(7) (pp. 3501−7).
2. Kalli, A., and Håkansson, K. (2010, September) “Electron capture dissociation of highly charged proteolytic peptides from Lys N, Lys C and Glu C digestion,” Molecular Biosystems, 6(9) (pp. 1668−81).
3. Cannon, J., et al. (2010) “High-throughput middle-down analysis using an Orbitrap,” Journal of Proteome Research, 9(8) (pp. 3886−90).
4. ROCCIT MS/MS Search Engine. (2013). Available at http://roccit.caltech.edu.
Post Author: Emily Humphreys. As an undergraduate studying biology at the University of Utah, Emily balanced a heavy class schedule while working long hours in a lab studying eye development. Following graduation, she became involved in infectious disease and aging research involving SNPS.
While she enjoyed the thrill of research, Emily has since traded bench work for science journalism.
She has spent the last year writing about new developments involved in proteomics research, and now food testing.
When she isn’t writing,she can be found playing outside with her kids.