Applications

Owned by Danielle Swaney
Last updated: Dec 12, 2018 by Robyn Kaake
3 min read

Protein-protein interaction (PPI) analysis

This tends to fall into a few different categories:

  • Affinity-Purification mass spectrometry (AP-MS):  Requires an affinity tagged bait (3xFLAG, Strep, GFP, etc.).
  • Endogenous proteins immunoprecipitation:  When you are only interested in identifying or quantifying interaction partners for a single protein and you have a good antibody for that protein.
  • Proximity-Labeling MS:  Relies on fusion of a labeling enzyme (APEX, BirA, etc.) to your protein of interest.  Labels everything in ~10-20nm proximity of your fusion protein, but direct binding proteins, as well as those only in spatial proximity.  Can be better for identifying transient interaction partners, at the cost of decreased specificity.

In any of these contexts experimental design is critical.  The average "purification" results in the the identification of 500-2000 proteins by MS analysis - the vast majority of which are background proteins binding to your beads.  As such, having controls that well represent your background is critical.  These can include one or several options, such as:

  • empty vector purification
  • GFP along
  • spatial reference proteins (i.e. proteins you expect to reside in similar cellular localization, but not within the same protein complex).


Global protein abundance and post-translational modification (PTM) profiling

  • When utilized:  When you are looking to quantitatively measure how protein or post-translational modifications (PTMs) are regulated under different conditions.  
  • Quantification approach:  Our method of choice is label-free quantification, which requires a minimum of 3 biological replicates per condition for sufficient statistical power.
  • PTMs we study:  Phosphorylation and ubiquitylation are the most commonly characterized PTMs, as robust enrichment approaches for these modification.  Less common are lysine acetylation, or phosphotyrosine.  
  • Requested amount of protein for analysis: 
    • protein abundance:  50ug/replicate
    • phosphorylation:  500-1000ug/replicate
    • ubiquitylation:  5-10mg/replicate


Quantitative targeted proteomics

  • When utilized: Targeted proteomics is a hypothesis driven method. We use it if you are interested in a specific set of proteins and you would like to quantify them accurately and consistently across a larger set of samples/various conditions.
  • Prior knowledge: To perform a targeted proteomics experiment prior knowledge of proteins of interest is required, e.g. from an initial global proteomics experiment to validate the findings or test these proteins across additional conditions. For each protein a targeted assay needs to be developed in advance, which is easiest to do from a pilot global proteomics experiment or information extracted from available literature and databases.
  • Number of proteins to be tested by targeted proteomics: For a high sensitivity the number of proteins to be targeted in one samples should be maximum 50-100 proteins.
  • Requested amount of protein for analysis: The amounts used for the targeted MS approach are the same than used for global approaches.


Crosslinking mass spectrometry (XL-MS)

Currently, our XL-MS applications fall into two different categories:

  • Affinity-Purification mass spectrometry (AP-MS):
    • When utilized: In cases where the target/prey interactions are believed to be transient or weak in nature, we use in cell chemical cross-linking to capture these interactions. 
    • Requirements: Requires an affinity tagged bait (3xFLAG, Strep, GFP, etc.); in some cases requires special tag/preparation techniques (i.e. TAP tags, HTBH tag, and denaturing conditions).
  • Structural XL-MS: 
    • When utilized: (1) Protein complex has flexible components; (2) is conformationally heterogeneous; (3) is too large, small, unstable, or impure for other structural techniques; and/or (4) to supplement other structural data for integrative modeling. 
    • Requirements: Protein components must be known and sequences provided; the complex must be stable in any non-amine containing buffer
    • Size of Protein/Complex: Any size or number of components
    • Requested amount of protein for analysis: NOTE that this will always be complex and size dependent
      • Standard In Gel Analyses: 100uL of 1-10uM sample/replicate
      • Standard In Solution Analyses: 20uL of 1-100uM sample/replicate

Targeted protein characterization

  • When utilized: When you are interested in characterizing PTMs on a specific protein.