DinosaurProteomicsDinosaur

Proteomics

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

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Dinosaur Proteins

First time such soft-tissue had been reported

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Many are skeptical.

MOR 1125 "B-rex"

• Discovered in 2000• 68-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.

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Tandem Mass-Spectrometry

• How much protein is needed?• Subpicomole? (10-12) Femtomole? (10-15)

• 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.

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Sample Preparation for Tandem Mass Spectrometry

Enzymatic Digestand

Fractionation

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Single Stage MS

MS

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Tandem Mass Spectrometry(MS/MS)

MS/MS

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Peptide Fragmentation

K1166

L1020

E907

D778

E663

E534

L405

F292

G145

S88 b ions

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0250 500 750 1000

m/z

% I

nte

nsit

y

147260389504633762875102210801166 y ions

y6

y7

y2 y3 y4

y5

y8 y9

b3

b5 b6 b7b8 b9

b4

Mastodon Peptide Matches

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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

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Mastodon Peptide Matches

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Tyrannosaurus Rex

• Very lowfmolepeptides

• Extra samplecleanuprequired.

• Some pretty low scores here…• Are these peptides statistical artifacts?

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Millions of Monkeys…

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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?

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+16 Amino-Acid Substitutions

A → SD → MF → YI → EL → E

M → FP → IP → LS → CV → D

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Confirmatory Study

• Seems to be silencing the critics…

• Much better job of the data-analysis

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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!

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