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Protein fragments sequenced in 68 million-year-old Tyrannosaurus rex

y said. "John of course took the bait." Schweitzer readily agreed to provide T. rex bone samples. "She knew from our last collaboration that I was not going to stop until I found something," Asara said.

At the outset, he faced two challenges. The first was to gather enough protein to sequence. The bone extract, sent by Schweitzer, arrived in the form of a gritty brown powder that had to be rid of contaminants. Using techniques and tricks perfected while working on the mammoth sample, Asara purified the protein, identified as collagen, and, with the enzyme trypsin, broke it down into fragments, or peptides, 10 to 20 amino acids long. The peptides were passed over a liquid chromatography (LC) column, where they were separated from one another and then sprayed at extremely low, or nanoliter, flow rates, for optimal sensitivity, into a mass spectrometer.

Typically, a mass spectrometer measures the mass, specifically the mass-to-charge ratio, of peptides as they come off the LC column. To maximize his yield, Asara used an ion trap mass spectrometer, which captures and holds peptides through time. The collected peptides were measured for mass and, in a second step, isolated and fragmented to reveal their amino acid sequence. Using this two-step, or tandem (MS/MS), procedure, Asara netted seven separate strings of amino acid.

He now faced his second challenge, namely, to interpret the amino acid sequences. Normally, when a sequence comes out of a mass spectrometer, it is compared to a database of existing amino acid sequences. Collagen is a highly conserved protein, so it was highly likely that some of the dinosaur peptide sequences would match those of an existing species. Of the seven T. rex peptides, five were for a particular class of collagen protein, collagen alpha I. The majority of these were found to be identical matches to amino acid sequences found in chicken collagen alpha I, while others matched newt and frog.

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Source:Harvard Medical School


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