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Max Planck PTM Studies Suggest Benefits of Manual Inspection of Mass Spec Data

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NEW YORK (GenomeWeb) – Advances in mass spec technology have steadily increased the breadth and throughput of proteomics experiments. In some cases, though, less may be more, suggested Ivan Matic, a researcher at the Max Planck Institute for Biology of Ageing.

Specifically, Matic said, careful manual inspection of MS/MS spectra can lead to new and unexpected observations, particularly with regard to protein post-translational modifications, his primary field of study.

Matic cited two recent studies from his group, one published this month in Cell and the other published this month in Nature Chemical Biology, that demonstrate the benefit of such an approach. In the first, he and his co-authors identified a new form of ubiquitination involved in Legionella pneumophila infections. In the second, they identified ADP-ribosylation as a novel type of histone modification that is involved in DNA damage response.

In both studies, the researchers' observations emerged in part from a focused, manual analysis of small amounts of mass spec data, Matic said, suggesting that such manual inspection "can really lead to completely unexpected and sometimes previously overlooked PTMs."

This, he noted, runs somewhat counter to the direction proteomics has trended and continues to trend.

"Technology is advancing and becoming more and more mature, not only mass spectrometry but also the software and algorithms that you use to analyze [mass spec] data," he said. "So, in general, mass spec is becoming something that many different people can use without having really advanced expertise."

This, Matic said, "is certainly a very good development," as it has broadened the use of proteomics approaches. At the same time, however, the fact that it is possible to do large-scale characterization of proteomes without looking at the raw MS/MS spectra means some things are likely going overlooked.

Analysis of mass spec proteomics data relies on a variety of assumptions, he noted. For instance, in the case of PTMs, "we assume the modifications have a specific mass, we assume that they occur on specific residues."

This, however, is not always the case, Matic said, adding that the exceptions to these assumptions can point towards new discoveries.

He cited as an example a 2014 Nature Communications study he and colleagues published in which they identified cysteine as a target for ADP-ribosylation on poly(adenosine diphosphate (ADP)-ribose) polymerase (PARP) proteins.

"I was looking for [ADP-ribosylation] on aspartate and glutamate, but then when I inspected a few MS/MS spectra I always saw a gap between what I thought were the modified residues and the cysteines," he said, noting that this led him to explore the possibility that the serines were ADP-ribosylated.

"It's not possible, of course, to look at 50,000 MS/MS spectra," he said. "But sometimes an inspection of a few MS/MS spectra can make you realize there is something weird there and maybe you should go back and think about what the modification really is."

Improving mass spec technology and fragmentation techniques allow researchers to gather more data than ever from MS/MS spectra, Matic noted.

"You have very high resolution at the MS and MS/MS level," he said, and different combinations of fragmentation methods allow researchers to look more closely at different parts of their target peptides.

In the case of serine ADP-ribosylation, for instance, use of electron-transfer dissociation allows researchers to localize the modification by fragmenting the peptide while leaving the PTM intact, Matic said. They can then follow this with collision-induced dissociation to look at the linkage between this modification and the substrate peptide.

In the Cell study, identification of the interaction of phosphoribosylated ubiquitin with serine residues on protein substrates suggests a potential process through which L. pneumophila exerts its pathogenic effects on mammalian hosts, Matic said.

In the Nature Chemical Biology study, identification of histone serine ADP-ribosylation suggested the possibility of a new marker for DNA damage. Upon exposing human osteosarcoma cells to oxidative DNA damage for 10 minutes, the researchers found that histone serine ADP-ribosylation increased between 10- and 27-fold. At the same time, they noted, "the canonical histone PTMs remained largely unchanged, except for two ubiquitination marks that increased slightly."

Once identified, such modifications can then be studied on a larger scale with higher throughput, Matic said. But "manual inspection is sometimes where the creative process can start. Because you can see something that is not really what you expected."

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