Posts Tagged ‘bottom-up’

A Protease for ‘Middle-down’ Proteomics

Author and Reporter: Ritu Saxena, Ph.D.

Neil Kelleher and his research team at Northwestern University have developed a method for enzymatic proteolysis large peptides for mass spectrometry–based proteomics using a protease OmpT. The method was published in a recent issue of the journal Nature.

Proteomics is defined as the study of the structure and function of proteins. Proteomic technologies will play an important role in drug discovery, diagnostics and molecular medicine because is the link between genes, proteins and disease. As researchers study defective proteins that cause particular diseases, their findings will help develop new drugs that either alter the shape of a defective protein or mimic a missing one. Proteomics, although refers to the study of the structure and function of proteins, it is often specifically used for protein purification and mass spectrometry.

‘Bottom-up’ and ‘Top-down’ are the two main strategies for proteomic studies using mass spectrometry. In Bottom-up proteomics referred to as the more common method, proteins are broken down into smaller pieces through enzymatic digestion followed by characterization into amino acid sequences and post translational modifications prior to analysis by mass spectrometry. By identifying and sequencing these smaller pieces, researchers can then determine the identity of the protein they make up. In Top-down proteomics, on the other hand, the process of proteolysis is skipped and it focuses on complete characterization of intact proteins and their post-translational modifications (PTMs).

“Although both the top-down and bottom-up approaches continue to mature, they each have limitations. The tryptic peptides used in the bottom-up approach are the primary unit of measurement, but their relatively small size (typically ~8–25 residues long) leads to problems such as sample complex­ity, difficulties in assigning peptides to specific gene products rather than protein groups, and loss of single and combinato­rial PTM information. The top-down approach handles these issues by characterizing intact proteins, but its success declines in the high-mass region. Therefore, a hybrid approach based on 2–20 kDa peptides could unite positive aspects of both bottom-up and top-down proteomics” says Kelleher et al in the research article.

The hybrid approach, referred to as ‘middle-down’ proteomics would enable the analysis of complex mixtures pre-sorted by protein size. Previously research efforts ‘middle-down’ proteomics included exploring the restricted proteolysis with enzyme alternatives to Trypsin and chemical methods (such as microwave-assisted acid hydrolysis), However, these methods generated peptides that were marginally longer than those produced by trypsin digestion. For the current study, Kelleher adds “We established an OmpT-based middle-down platform to analyze complex mixtures pre-sorted by protein size. After inte­grating the data from the middle-down workflow that was applied to ~20–100-kDa proteins fractionated from the HeLa cell proteome, we identified 3,697 unique peptides (average size: 6.3 kDa) from 1,038 unique proteins (26% average sequence coverage) at an esti­mated 1% false discovery rate”.

OmpT, a protease derived from Escherichia coli K12 outer membrane belongs to the novel omptin protease family10 and is known to cleave between two consecutive basic amino acid residues (Lys/Arg-Lys/Arg). The authors developed OmpT into an efficient rea­gent to generate >2-kDa peptides for middle-down proteomics, thus, utilizing OmpT to achieve robust, yet restricted, proteolysis of a complex genome.

Researcher Kelleher and his team have been in news earlier for their work on ‘top-down’ proteomics when his team developed a new method that could separate and identify thousands of protein molecules quickly. In the first large-scale demonstration of the top-down method, the researchers were able to identify more than 3,000 protein forms created from 1,043 genes from human HeLa cells. The study was published in last year in the October issue of the journal Nature.

Thus, Kelleher and his group was able to demonstrate that OmpT-based proteomic approach has a robust and restricted proteolysis capacity making it an attractive option for mass-spectrometry-based analysis of primary structure of protein.


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