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• Circadian rhythms refer to cycles that revolve
around our days (~24 hours)
• This circadian clock regulates many biological
functions – from sleep to cellular metabolism
• This circadian clock is present even in the
absence of light, showing the importance in
regulation of cell function in our days
Artwork by Emma Darvick - https://giphy.com/gifs/illustration-sunset-sunrise-3o6ZsWSWIkZYHLHTyM
• Phosphorylation is a reversible, post-
translational method of altering proteins
• Phosphorylation occurs from the binding of a
phosphate group to an amino acid
• These modifications can cause proteins to be
activated, deactivated, or even modify their
function in the cell
Gla086 [CC BY-SA 3.0 (https://creativecommons.org/licenses/by-sa/3.0)]
Serine
Phosphorylated Serine
• The biological makeup of the ‘core clock’ is
conserved between fungi and animals
• There is a transcription-translation negative
feedback loop between a heterodimer complex
that can be defined as a positive arm and a
negative arm
• The positive arm influences genes unrelated to
this core clock mechanism – these genes are
dubbed clock controlled genes (ccgs)
• The positive arm is comprised of WC-1/WC-2
proteins
• The negative arm is comprised of FRQ/FRH
proteins
• The many small sources of variation from non-
biological sources, e.g. different reagents and
handlers, are known as batch effects
• Batch effects likely influence all experiments
but are of greater effect in high-throughput
analyses like proteomics
• LIMBR, a python package designed for
circadian proteomics data, was used to
control for batch effects
• A positive value of γ indicates damping, or
decreasing amplitude over time
• A negative value of γ indicates forcing or
increasing amplitude over time
• When γ = 0, the data is considered in truly
harmonic oscillation
Pattern Legend
A = damped
B = forced
C = harmonic
D = overexpressed
E = repressed Hannah De los Santos et. al, ECHO: an Application for Detection and Analysis of Oscillators Identifies Metabolic Regulation
on Genome-Wide Circadian Output, Bioinformatics, , btz617, https://doi.org/10.1093/bioinformatics/btz617
Phosphopeptide Expression over 48 Hours
– All ECHO data
• Expressions are sorted from top to bottom by
phase and normalized to be within the range
[-1,1]
• Each column represents a 2-hour interval
• Our oscillatory data can be classified into
three groups by their amplitude change
coefficient (γ)
• Total ECHO expressions: 255
Phosphopeptide Expression over 48 Hours
– Harmonic
• Harmonic classified data (−0.03 ≤ γ ≤0.03)
ebbs and flows consistently over time
• Total Harmonic expressions: 70
Phosphopeptide Expression over 48 Hours
– Forced
• Forced classified data (−0.15 ≤ γ ≤ −0.03)
increases in amplitude over time
• Total Forced expressions: 79
Phosphopeptide Expression over 48 Hours
– Damped
• Damped classified data (0.15 ≤ γ ≤ 0.03)
increases in amplitude over time
• Total Damped expressions: 55
• Investigate the ECHO data for
regulatory proteins and any relations
to the core clock function
• FRQ protein was found in the ECHO
data and classified as overexpressed
• Identify specific phosphorylated
amino acids that may be key in
circadian regulation
- FRQ
Circadian Rhythms in Phosphoproteomics

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Circadian Rhythms in Phosphoproteomics

  • 1.
  • 4. • Circadian rhythms refer to cycles that revolve around our days (~24 hours) • This circadian clock regulates many biological functions – from sleep to cellular metabolism • This circadian clock is present even in the absence of light, showing the importance in regulation of cell function in our days Artwork by Emma Darvick - https://giphy.com/gifs/illustration-sunset-sunrise-3o6ZsWSWIkZYHLHTyM
  • 5. • Phosphorylation is a reversible, post- translational method of altering proteins • Phosphorylation occurs from the binding of a phosphate group to an amino acid • These modifications can cause proteins to be activated, deactivated, or even modify their function in the cell Gla086 [CC BY-SA 3.0 (https://creativecommons.org/licenses/by-sa/3.0)] Serine Phosphorylated Serine
  • 6. • The biological makeup of the ‘core clock’ is conserved between fungi and animals • There is a transcription-translation negative feedback loop between a heterodimer complex that can be defined as a positive arm and a negative arm • The positive arm influences genes unrelated to this core clock mechanism – these genes are dubbed clock controlled genes (ccgs) • The positive arm is comprised of WC-1/WC-2 proteins • The negative arm is comprised of FRQ/FRH proteins
  • 7.
  • 8. • The many small sources of variation from non- biological sources, e.g. different reagents and handlers, are known as batch effects • Batch effects likely influence all experiments but are of greater effect in high-throughput analyses like proteomics • LIMBR, a python package designed for circadian proteomics data, was used to control for batch effects
  • 9. • A positive value of γ indicates damping, or decreasing amplitude over time • A negative value of γ indicates forcing or increasing amplitude over time • When γ = 0, the data is considered in truly harmonic oscillation Pattern Legend A = damped B = forced C = harmonic D = overexpressed E = repressed Hannah De los Santos et. al, ECHO: an Application for Detection and Analysis of Oscillators Identifies Metabolic Regulation on Genome-Wide Circadian Output, Bioinformatics, , btz617, https://doi.org/10.1093/bioinformatics/btz617
  • 10. Phosphopeptide Expression over 48 Hours – All ECHO data • Expressions are sorted from top to bottom by phase and normalized to be within the range [-1,1] • Each column represents a 2-hour interval • Our oscillatory data can be classified into three groups by their amplitude change coefficient (γ) • Total ECHO expressions: 255
  • 11. Phosphopeptide Expression over 48 Hours – Harmonic • Harmonic classified data (−0.03 ≤ γ ≤0.03) ebbs and flows consistently over time • Total Harmonic expressions: 70
  • 12. Phosphopeptide Expression over 48 Hours – Forced • Forced classified data (−0.15 ≤ γ ≤ −0.03) increases in amplitude over time • Total Forced expressions: 79
  • 13. Phosphopeptide Expression over 48 Hours – Damped • Damped classified data (0.15 ≤ γ ≤ 0.03) increases in amplitude over time • Total Damped expressions: 55
  • 14. • Investigate the ECHO data for regulatory proteins and any relations to the core clock function • FRQ protein was found in the ECHO data and classified as overexpressed • Identify specific phosphorylated amino acids that may be key in circadian regulation - FRQ