NEW YORK (GenomeWeb) – University of Texas researchers have developed a new mass spec workflow that could help enable more effective middle-down proteomic experiments.
Detailed in a paper published last month in Analytical Chemistry, the method combines carbamylation of proteins prior to trypsin digestion to increase the average size of digested peptides with ultraviolet photodissociation (UVPD) mass spec to improve their sequence coverage.
The peptides generated by the method still fall short of what would be an ideal length for middle-down experiments, noted Jennifer Brodbelt, professor of chemistry at UT and senior author on the paper. However, the carbamylation approach did generate substantially longer peptides than did standard trypsin digestion, and UVPD proved more effective than more conventional fragmentation approaches for sequencing these peptides.
Middle-down proteomics aims to stake out ground between conventional bottom-up approaches and the less widespread but growing practice of top-down, or intact protein, proteomic analysis. The argument for the approach is that it would achieve some of the top-down community's goals of improving study of protein isoforms and post-translational modifications while avoiding the technical challenges inherent in measuring intact proteins on a proteome-wide scale. Larger peptides would also reduce the complexity of peptide digests being studied, which could improve analyses.
Middle-down efforts have thus far run into the problem that there is no obvious way to reproducibly digest proteins in the 4 kDa to 10 kDA range that Brodbelt said would be ideal for middle-down assays. Trypsin digestion, which is commonly used in bottom-up workflows, generally produces peptides of around 1 kDa.
In previous studies, researchers have tried various approaches to generating the longer peptides required for middle-down work. For instance, earlier this year, Utrecht University's Albert Heck published on efforts using both the proteases Asp-N and Glu-C, as well as a non-enzymatic method in which they incubated proteins at high temperatures in diluted formic acid. The protease-based method generated peptides with average size of 1.5 kDa, while the formic acid method generated average peptide sizes of 1.9 kDA.
Another route researchers have pursued is using the outer membrane protease T, OmpT. OmpT is a di-basic protease, meaning that it recognizes not one but two amino acids and will cut only at sites where both are present, which typically leads to production of longer peptides compared to trypsin. However, the enzyme is not entirely specific for di-basic sites. Additionally, not all proteins have di-basic sites, meaning experiments using it won't be able to achieve full proteome coverage.
In their recent study, Brodbelt and her colleagues used trypsin for digestion, but prior to digestion they subjected their samples to carbamylation, which prevents trypsin from cleaving peptides at lysine residues, limiting cleavage to arginine residues, which are typically less common.
Applying this approach to Escherichia coli samples, they generated peptides with an average size of 2.2 kDA, with 18 percent of generated peptides larger than 3 kDa and a small proportion in the 6 kDA and above range.
This matched fairly well to the peptide size distribution predicted by an in silico digest, which produced peptides with an average mass of 2.3 kDA with 25 percent of peptides having a mass of 3 kDa or greater.
The data represents an experiment using UVPD-based fractionation, which Brodbelt noted, "provides more extensive coverage of longer peptides," compared to commonly used fragmentation approaches like higher-energy collisional dissociation (HCD). This, she suggested, makes UVPD a promising approach for middle-down analysis. Using HCD to analyze the same carbamylated E. coli digest, the researchers found an average peptide size of 2 kDa with 11 percent of peptides greater than 3 kDa in size.
The difference between the UVPD and HCD peptide repertoires reflects the fact that UVPD provides superior sequence coverage of longer peptides, allowing for more long peptides to be confidently identified, the researchers noted. Similarly, they observed that a comparison of standard trypsin digestion and the carbamylation-trypsin digestion found that carbamylation provided "a negligible difference" in sequence coverage when using HCD, but that in the UVPD experiments around 75 percent of proteins showed increased sequence coverage using the carbamylation-trypsin approach.
UVPD uses photons to excite and fragment ions for mass spec analysis. The approach, which is one of the primary research interests of Brodbelt's lab, is not as widely used in proteomics as other fragmentation methods, but has shown promise particularly for analysis of larger peptides and intact proteins.
"For small peptides like standard tryptic peptides, HCD works tremendously well, and it is hard to beat," Brodbelt said. "However, for increasingly large peptides, UVPD can and will out-perform HCD. UVPD will provide more extensive coverage of the longer peptides, and that contributes to better characterization of the protein sequences."
While the carbamylation-trypsin digestion approach still falls short of the field's goals for middle-down proteomics, Brodbelt suggested it could still prove useful combined with UVPD mass spec.
"I would envision using the carbamylation-UVPD approach in parallel with standard trypsin-based bottom-up strategies," she said. "It would be good for cases where one really needs to confidently characterize sequences across regions where there might be mutations that could be overlooked or missed by patchy bottom-up methods."
The researchers also compared the carbamylation-trypsin approach to digests using the protease Arg-C. Like the carbamylation-trypsin method, Arg-C cleaves proteins at arginine residues, meaning the two should produce similar digests. However, Arg-C has been shown to cleave non-specifically, as well. Their analysis bore this out, finding that in Arc-C-treated samples 80 percent of peptides resulted from cleavage at an arginine, with the remaining 20 percent resulting from cleavage at some other residue. In the carbamylation-trypsin-treated samples 91 percent of peptides were produced by cleavage at an arginine.
Brodbelt said that she and her colleagues are continuing to explore methods for consistently producing longer peptides for middle-down analysis, looking primarily at ways to modify the behavior of commonly used proteases like trypsin.