CB 399: An Overview of Mass-Spectrometry-based Proteomics Spring 2013

An Overview of Mass-Spectrometry-based Proteomics
Nanocourse Directors: Steve Gygi and Jarrod Marto

Lecturers: Steve Gygi, Jarrod Marto, and Mathew Sowa
Curriculum Fellow: Jason Heustis, ronald_heustis@hms.harvard.edu

Mass spectrometry is the enabling tool in the field of proteomics. The field has advanced at such a pace that many papers now report the simultaneous identification of thousands of proteins directly from cells or tissues. We will discuss three areas where mass spectrometry can directly impact cell biology research. 1) Practical aspects of protein sequencing via mass spectrometry. 2) Determining protein-protein interactions by affinity purification coupled to mass spectrometry. 3) The use of stable isotopes to encode whole proteomes for protein profiling of different cell states.


First Session: Monday, April 8, 2013 1 PM – 4:30 PM
Location: Armenise Amphitheater
Second Session: Monday, April 15, 2013 1PM – 3:30 PM
Location: TMEC Building, Room 209, followed by tour


DROP DEADLINE: Monday, April 1, 2013


Recommended background readings: (log on below to download)



Domon, B and Aebersold, R. (2006)  Mass Spectrometry and Protein Analysis.  Science, vol:312, pp. 213 – 217.

Bantscheff, M, et al.  (2007)  Quantitative mass spectrometry in proteomics: a critical review. Anal Bioanal Chem, vol:389, pp.1017 – 1031.

Dunham, WH, Mullin, M, Gingras, AC.  (2012) Affinity-purification coupled to mass spectrometry: Basic principles and strategies.  Proteomics. vol:12, pp. 1576 – 1590.

Nesvizhskii AI. (2012)  Computational and informatics strategies for identification of specific protein interaction partners in affinity purification mass spectrometry experiments.  Proteomics. vol:12, 1639 – 1655.

Primary Articles

Kratchmarova, I, et al. (2005)  Mechanism of Divergent Growth Factor Effects in Mesenchymal Stem Cell Differentiation.  Science, vol:308, pp. 1472 – 1477.


DMS students may view a video presentation of the Day 1 lectures online.  In order to view instructions guiding you to this video, please log into this website and access the file posted along with readings and assignments.


Required Assignment for second session: (log on below to download)

Questions for Discussion

Monday, April 15th, 2013, 1:00 pm – 3:30 pm

Room TMEC 209


Instructions:  Please respond by providing a one-paragraph response to each question.  Your assignment will be turned in.

  1. What is the difference between “top-down” and “bottom-up” proteomics?
  2. How does a mass spectrometer “sequence” a protein? How does an algorithm like Sequest identify sequence from tandem mass spectra?  How does it identify phosphorylated peptides?
  3. How many proteins are expressed in a HeLa cell or a yeast cell?  How many proteins can large-scale proteomics experiments identify?  How big is this gap?
  4. Distinguish between absolute and relative quantification and how they are typically used in proteomics studies
  5. Discuss current limitations in interpretation of large-scale proteomics data sets.
  6. After identifying putative protein-protein interactions using an AP-MS/MS approach, describe at least 3 validation experiments with specific attention to each's advantages and disadvantages. 
  7. What are the advantages/disadvantages of using epitope tagged proteins for AP-MS/MS experiments versus endogenous IP-MS/MS analysis? How might the disadvantages of an endogenous IP-MS/MS experiment be mitigated?