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Guest Lecture
KIN 225
Brenna Hay, PhD(c)
University of British Columbia
March 20, 2023
Proteins are super important!
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Central dogma of biology:
○ DNA à RNA à protein
Proteins mediate virtually every process
that takes place in a cell
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Amino Acid Structure
H
R
O
C
C
O
-
+
NH3
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Amino acid à peptide à protein
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Protein degradation
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Via oxidative degradation
During:
○ Normal synthesis and degradation of
cellular proteins (protein turnover)
○ Times of dietary excess of protein
(ingested amino acids > body’s needs)
○ Times of fasting/starvation or during
very prolonged exercise
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Protein degradation: glucose
alanine cycle
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Activated during fasting/starvation or
during very prolonged exercise
Purpose: to maintain blood glucose levels
○ Degrades muscle protein to produce
glucose (to generate additional ATP)
May provide 5-10% of energy needs during
prolonged exercise
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Amino Acid Structure
H
R
O
C
C
O
-
+
NH3
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Amino Acid Structure: Alanine
H
CH3
O
C
C
-
O
+
NH3
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Glucose alanine cycle
Ishikura et al., 2013
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Protein as a fuel source
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Proteins are digested into amino acids
Usable forms of amino acids can be used
either directly in muscle or by conversion
to glucose (gluconeogenesis) in liver
Amine group (NH3) must be removed first
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Amino Acid Structure
H
R
O
C
C
O
-
+
NH3
“Amino group”
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Removal of NH3
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▷
Remove NH3 to break it down into the usable form of
the amino ACID
Removal of NH3 by:
○ Transamination (transferring the amine)
○ Oxidative deamination (removing the amine)
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Removal of NH3
1) “Transamination”:
▷ Exchange of NH3 between molecules
- Nitrogen removed and passed to another compound to be used again
▷ Transaminase (enzyme) involved
▷ Mainly in muscle
Amino Acid
not usable
NH +
3
Amine
Acceptor
Transaminase
Amino Acid
Amine
Acceptor
NH3+
usable
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Removal of NH3
2) “Oxidative deamination”
▷ Removal of NH3
▷ N excreted from the body
- Filtered through kidney and eliminated through urine
- Increases bodies need for water
▷ Mainly in the liver
▷ Deaminase (enzyme) involved
▷ Convert deaminated amino acids to pyruvate or acetyl coA
- Enter citric acid cycle for oxidation
Amino Acid
not usable
NH +
3
Deaminase
Amino Acid
usable
NH3+
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Protein as a fuel source
Amino acids can be:
▷ Glucogenic (can convert to glucose)
○ When deaminated form
■ Pyruvate
■ Oxaloacetate
■ Malate
▷
Ketogenic (can convert to ketone bodies)
○ When deaminated form
■ Acetyl-CoA (for Citric Acid Cycle)
■ Acetoacetate
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Common pathway
▷ After NH3 is removed, carbon skeleton can enter
the CAC / Krebs cycle
Pyruvate
NAD+
NADH
NAD+
Acetyl CoA
NADH
Krebs Cycle
NAD+
FAD
FAD H
ADP
ATP
NAD+
NADH
NADH
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Importance of amino acids during
prolonged aerobic exercise or starvation
▷
Replenishment of intermediates of the
Krebs Cycle
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Amino acids can be converted to glucose in
the liver. This glucose can then go back to
the muscle to be used as a fuel source
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The ‘omics revolution
CISN
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Systems biology
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Proteomics & the study of proteins
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Proteome = the complete set of proteins
made by an organism
How can we study the proteome?
○ Mass spectrometry!
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Biorender
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How can we apply mass spectrometry
to real-world problems?
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Choose your own adventure…
○ A forensic investigation of snake venom
○ Anti-doping in sport
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Forensic investigation of snake venom
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The investigation:
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Could there be snake venom in the blood?
○ Mass spectrometry will tell us!
Prepared a blood sample for mass
spectrometry
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The investigation: challenges
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There were LOTS of blood proteins that
overwhelmed the signal of potential snake
proteins
○ Solution: fractionation and immunodepletion
A lot of human and snake proteins look quite
similar
○ Solution: deep bioinformatics search and
manual inspection of potential snake proteins
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The outcome
▷ <1% of the sequenced peptides were
matched unequivocally to snake proteins
▷ Only 1 (of 111) snake peptides matched to a
well-known snake venom protein,
phospholipase A2
▷ A follow-up comparison validated the
presence of snake peptides in snake venom
▷ Additional DNA validation was performed by
a second lab and confirmed these results
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The outcome
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This evidence, in part, led to a court-issued
search warrant of the defendant’s home,
followed by his arrest and an eventual
guilty plea with formal sentencing to 18
months in prison.
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Mass spectrometry for anti-doping
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Why do we ban substances in organized
sports?
Gold standard: World Anti-Doping Agency
How do they test for banned substances?
○ Mass spectrometry! (and NMR)
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Anabolic agents
Peptide hormones, growth factors, and
mimetics
Beta-2 agonists
Hormone and metabolic modulators
Diuretics and masking agents
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Anabolic agents
Peptide hormones,
growth
factors, and
Are these
proteins?
mimetics
If not, how can mass spectrometry
Beta-2 agonists
detect them?
Hormone and metabolic modulators
Diuretics and masking agents
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Considerations for metabolomics
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More complex than proteomics (MANY
small molecules in any given class
compared to only 20 amino acids)
You can’t detect what you don’t know
exists
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Icarus: truth is the new banned substance
▷ Bryan Fogel documents his exploration of
doping to win an amateur cycling race
▷ In doing so, he meets Grigory Rodchenkov,
the former head of Russia’s national antidoping lab
Icarus
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40 publications on PubMed!
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We can extend the detection time of
banned GHRPs by also looking for
presence of key metabolites in urine
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How can they beat the mass spec?
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They couldn’t!
Sample bottles
were emptied,
cleaned, and refilled with
athlete’s untainted
urine collected
previously
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Thank you!
Foster Lab
Jan Lab
Committee:
Nathan Louie
Daniel Andrews
Dr. Honglin Luo
Jenny Moon
Dr. Eric Jan
Dr. Thibault Mayor
Jason Rogalski
Past Lab Members
Funding
Jeanne Yuan
Dr. Craig Kerr
B.C. Graduate Scholarship
Dr. Leonard Foster
Dr. R Greg Stacey
NSERC
Renata Moravcová
CIHR
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