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ETH Team Uses Shotgun, Targeted Mass Spec to Study Protein Factors Involved in Ribosome Development

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Researchers at the Swiss Federal Institute of Technology Zurich have performed a targeted proteomic analysis of pre-ribosomal complexes using a combination of shotgun and selected-reaction monitoring mass spectrometry.

Detailed in a paper published this week in Molecular Systems Biology, the study identified several hundred factors involved in construction and localization of pre-ribosomal particles in yeast and quantified 50 of them across different points in the process.

The study identified several assembly factors that accompany pre-60S pre-ribosomal particles – the larger of the two subunits that make up a ribosome – to the cytoplasm, including a novel shuttling factor involved in nuclear transport. Additionally, it demonstrated the potential of combined discovery and targeted mass spec approaches for unraveling biological systems, Vikram Panse, an ETH researcher and leader of the project, told ProteoMonitor.

Construction of eukaryotic ribosomes is a complicated process involving a large number of protein factors for assembly and intracellular transport of these complexes. Previous proteomic studies have helped scientists catalogue many of the factors involved, but, Panse said, recent advances in mass spectrometry have enabled both more comprehensive and quantitative study of these proteins, offering new insights into their function.

The first shotgun mass spec analysis of pre-60S pre-ribsosomal particles was done over a decade ago and identified around 200 factors involved, Panse noted. On the other hand, the analysis in the MSB study, which was led by ETH researcher Paola Picotti, identified more than 300 proteins, Panse said.

The ETH researchers used an AB Sciex TripleTOF 5600 instrument for the shotgun portion of their experiment. Then, using the data generated via that analysis they built SRM assays for 50 of the identified proteins, which they then quantified using an AB Sciex 5500 QTrap.

"In the first stage we did a very comprehensive shotgun analysis just to see what kind of peptides we were seeing in the reaction mixture," Panse said. With that information, the researchers were able to identify peptides that looked like promising targets for SRM analysis.

The researchers limited their targeted analysis to 50 proteins because they felt that approaches the current technical ceiling of SRM multiplexing "in realistic terms," Panse said. He noted that they were stringent in their assay standards, requiring identification of five peptides per protein and three to five transitions per peptide. They were able to quantify their 50 targets in a single mass spec run of roughly one hour.

The data generated by the ETH team's shotgun analysis should give other researchers raw material with which to develop SRM assays to their own proteins of interest, Panse said. "We've given them the best flying peptides with which to develop these assays, so they should be able to do it now with any of these hundreds of proteins."

Panse's ETH colleague Ruedi Aebersold has been working with the TripleTOF to develop a data-independent acquisition technique capable of SRM-style quantitation of all the peptides in a given sample (PM 1/13/2012). In theory, this technique could have allowed the researchers to quantify all 300-plus proteins identified in their initial shotgun experiment as opposed to the subset of 50. However, Panse said, while SWATH "has great promise," he felt it was "not as sensitive or precise yet as I want my biology to be."

"We are not a proteomics labs, we're really a biology lab," he said. "So I think we had to have this kind of balance. And so I decided, let's look at these 50 [proteins] very accurately [using standard SRM], and then we will move on to the next 50."

To obtain snapshots of the proteins involved in ribosome development at different points of the process, the researchers used two main approaches -- affinity purification of proteins associated with particular stages of development and genetic trapping, a technique that halts ribosome development by mutating genes coding for proteins key to the progression of the process.

"If you look at the [ribosome development] pathway, there are diverse enzymes – GTPases, ATPases – that remodel the ribosomes as they mature," Panse said. "In principal, you can create dominant negative mutants of all these different enzymes to block [the process] at different stages" and look at the different protein present at each stage.

In the MSB study, Panse and his colleagues created a dominant negative mutant of AAA-ATPase Drg1, an enzyme required for the first maturation step of pre-60s pre-ribsosomal particles. In the future, he said, they hope to create similar mutants to halt the process at a variety of stages.

In addition, he said, "we want to develop SRM assays for as many factors as possible – probably around 200 more – and develop protocols with which we can monitor all 200 at a time, which is a big technical challenge."

The approach used in the paper should be applicable to the study of any number of large complexes, Panse said. "This is a whole field in and of itself – how these big [complexes] are constructed and disassembled, and in principle SRM can be used for that purpose."

He added that while many biologists are still committed to traditional protein analysis approaches like Westerns and immunoassays, he sees mass spec, and mass spec for targeted protein quantitation in particular, making inroads into the field.

Mass spec-based proteomics "is still a new technology… and there are not enough people trained," Panse said, noting that even in the case of his lab, which has access to first-class proteomics technology via Aebersold, integrating the techniques into their work has taken time.

However, while SRM-MS "is not trivial yet," he predicted that it would soon become trivial, with machines incorporating catalogues of assays available to researchers at essentially the touch of a button.

"This is just the beginning," he said.

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