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Proteomics Introduction

General concept, LC-MS, DEP analysis

Korea University College of Medicine

Chembiol Lee group �(https://leegroup.chembiol.re.kr/home)

​

Eunteg Ryu

Jiwon Kim

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Proteome

1. What is Proteomics?

Large scale study of proteome, or whole set of proteins in given sample.

NOT one or few proteins

Protein expression

(Identification & quantification)

Protein interaction

Protein structure

Proteomics’ scope covers expression, interaction, and structure of proteins

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2. LC-MS

Many proteomics studies use MS (Mass Spectrometer), �often in combinataion with LC (Liquid Chromatography)

LC: for separation of proteins

MS: for analysis (identification, quantification) of proteins

* Mass analyzer in picture: Orbitrap

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3. Structure & Basic Principle of MS

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3. Structure & Basic Principle of MS

Mass spectrometer measure M/Z of an gas phase ion.

* M: mass of ion, Z: charge of ion

What does MS do?

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3. Structure & Basic Principle of MS

Structure of �Mass Spectrometer

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  • Ion source
  • Mass analyzer
  • Detector

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3. Structure & Basic Principle of MS

Ion source

: Gives charge to protein, and evaporates it.

Types of ion sources

    • ESI (Electrospray Ionization)
    • MALDI (matrix assisted laser desorption ionization)

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3. Structure & Basic Principle of MS

Mass analyzer

  • Separates ions according to M/Z.
  • Types of mass analyzer:�• Quadrupole mass analyzer�• Ion trap mass analyzer�• Time of flight�• Orbitrap (In the picture!)

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Detector

  • observe / collect separated ions

<Quadrupole mass analyzer>

https://www.chm.bris.ac.uk/ms/quadrupole.xhtml

<Quadrupole Ion Trap mass analyzer>

https://www.shimadzu.com/an/service-support/technical-support/analysis-basics/fundamental/mass_analyzers.html

https://www.shimadzu.com/an/service-support/technical-support/analysis-basics/fundamental/mass_analyzers.html

<Time of Flight mass analyzer>

https://www.shimadzu.com/an/service-support/technical-support/analysis-basics/fundamental/mass_analyzers.html

<Orbitrap mass analyzer>

Savaryn, John & Toby, Timothy & Kelleher, Neil. (2016). A researcher's guide to mass spectrometry‐based proteomics. PROTEOMICS. 16. 10.1002/pmic.201600113.

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4. MS/MS

(‘tandem mass’ or MS-N)

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4. MS/MS

MS-1 spectrum

m/z

https://en.wikipedia.org/wiki/Liquid_chromatography%E2%80%93mass_spectrometry

  1. Fragmentation of ion with certain m/z value
  2. Analyze with MS again

More information (especially, amino acid sequence) about protein of interest

https://depts.washington.edu/yeastrc/detection-and-quantitation-of-proteins-by-mass-spectrometry/

MS-2 spectrum

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5. Label-free / Label-baed quantification

How to compare protein expression profile in different samples(conditions)?

ex.

In healthy individual

In patient

Conclusion:

loss of

is related to disease

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5. Label-free / Label-baed quantification

https://www.uoguelph.ca/aac/facilities/mass-spectrometry-facility/quantitative-proteomics/quantitative-proteomics-label-or-not

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5. Label-free / Label-baed quantification

SILAC

(stable isotope label by amino acids in cell culture )

Culture cell in media containing different isotopes

Resulting samples contain same protein with different mass

Abundance of a protein in two condition can be directly compared in MS-1 spectra.

https://en.wikipedia.org/wiki/Stable_isotope_labeling_by_amino_acids_in_cell_culture

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5. Label-free / Label-baed quantification

TMT

(tandem mass tag)

https://www.researchgate.net/figure/QUANTITY-isobaric-tandem-mass-tags-for-glycan-labeling-and-quantitation-a-Molecular_fig2_285362063

Structure of Tandem Mass Tag

  • Different mass tags have same mass
  • They contain reporter group of different mass
    • Reporter group is released after fragmentation
    • Thus, different tags can be differentiated in MS-2 or MS-3.

ACS Infect. Dis. 2019, 5, 12, 2136–2147

Publication Date:October 10, 2019

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5. Label-free / Label-baed quantification

https://www.uoguelph.ca/aac/facilities/mass-spectrometry-facility/quantitative-proteomics/quantitative-proteomics-label-or-not

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6. Data analysis

Chen, C., Hou, J., Tanner, J. J., & Cheng, J. (2020). Bioinformatics Methods for Mass Spectrometry-Based Proteomics Data Analysis. International Journal of Molecular Sciences, 21(8). https://doi-org-ssl.oca.korea.ac.kr/10.3390/ijms21082873

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7. DEP analysis overview

Raw

Data

Maxquant

protein identification & quantification

What we are going to do!

created by Arne H. Smits

Jürgen Cox group, Max Plank institute of biochemistry