Dinosaur Proteomics

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Claims Proteins can be extracted from fossilized bones Extracted proteins can be analyzed by LC-MS/MS MS/MS can be matched to peptide sequences from modern species* Matched peptides offer insight on evolution of extinct species 3

Dinosaur Proteins First time such soft-tissue had been reported 4 Many are skeptical.

MOR 1125 "B-rex" Discovered in million years old Soft-tissue observed in femur Collagen is durable, resistant to degradation, abundant in bones, and highly conserved. Biochemical analysis is consistent with collagen Significant hydroxylation. 5

Tandem Mass-Spectrometry How much protein is needed? Subpicomole? ( ) Femtomole? ( ) What peptides should the spectra be matched to? No T. rex genomes around, but… Collagen is highly conserved, so match to modern collagen protein sequences Demonstrate first with ostrich and Mastodon, then T. rex. 6

7 Sample Preparation for Tandem Mass Spectrometry Enzymatic Digest and Fractionation

8 Single Stage MS MS

9 Tandem Mass Spectrometry (MS/MS) MS/MS

10 Peptide Fragmentation

Mastodon Peptide Matches 11

Generating novel peptides Use modern collagen sequences to propose novel peptides Voting heuristic, PAM (blast) matrix Only makes sense if changed amino- acid residues are close to each other 12

Mastodon Peptide Matches 13

Tyrannosaurus Rex Very low fmole peptides Extra sample cleanup required. Some pretty low scores here… Are these peptides statistical artifacts? 14

Millions of Monkeys… 15

Concerns Most of the peptide scores are weak… …with no way to correct for multiple testing Liberal use of hydroxylation (+16) mod… …increases false positives significantly Other expected modifications not checked… …deamidation is expected by others Collagen is highly conserved… …so many very similar peptides considered, …and few mutated amino-acids Contaminating collagen samples? Ostrich? Available sequences are poorly sampled Chicken, frog, newt? 16

+16 Amino-Acid Substitutions A → S D → M F → Y I → E L → E M → F P → I P → L S → C V → D 17

Confirmatory Study Seems to be silencing the critics… Much better job of the data-analysis 18

Lessons If you are publishing a controversial result, be conservative with your claims Poor statistical rigor can scuttle otherwise good work Peptide identification can be carried out from related amino-acid sequences… …but be careful, the devil is in the details. If you want a paper in Science, dig up some T. rex bones! 19