Three Decades of Experience in
Protein Contract Research

2D Mass Spectrometry for Proteoforms

  • First Step

    Protein samples are separated by 2D SDS PAGE

  • Second Step

    Proteoforms of interest are found with 2D WB and/or computer comparison of 2D patterns

  • Third Step

    Proteoform spots are cut from the 2D gel, trypsin digested for bottom-up analysis

  • Final Step

    Standard and custom database searches are performed

 

2D Gel Service Options

First Dimension: isoelectric focusing (IEF)

  • Standard IEF pH range 3-9; inquire about narrower ranges.

Second Dimension: Sodium dodecyl sulfate polyacrylamide (SDS PAGE)

  • Slab gel sizes: large format (22 x 20 cm) and standard format (13 x 15 cm).
  • Molecular weight range of 10-220 kDa for 10% acrylamide tris-glycine gels
  • 5% acrylamide tris-tricine gels used to detect smaller peptides down to 3 kDa

Slab Gel Staining Options

  • Standard: Silver and Coomassie blue staining
  • Sypro Ruby or Cy Dyes
  • Western blotting for detection of specific proteins or PTMs assuming antibodies are available.
  • Digitized 2D gel patterns are compared and using SpotMap software from TotalLab with optional quantification. Manual comparisons by eye are offered for faster detection of differences between two or more samples.

Additional Mass Spectrometry Services

  • Custom database search: we can custom search for any protein, for any possible proteoform, and for any possible PTM on that protein.
  • Characterization of the overexpressed proteins (in bacteria, yeast, insects, or mammalian cells): the proteins can be analyzed for impurities such as protein contaminants (host cell proteins), alternative N-terminus (with or without Methionine), PTMs, etc.
  • Protein-protein interactions (PPIs): both stable and transient protein-protein interaction can be identified by co-immunopurification (co-IP, i.e., immunopurification/IP using one antibody against protein 1, followed by western blotting using against protein 2. Alternatively, one could do IP using one antibody against protein 1, followed by SDS-PAGE and nanoLC-MS/MS analysis for identification of protein 2 (or more proteins). PPIs can also be identified by sample separation with BN-PAGE, followed by 1) western using the antibodies of interest or 2) Coomassie staining, followed by nanoLC-MS/MS analysis for identification of both transient or stable PPIs

    • Experienced Protein Biochemists

  • Kendrick Labs has been performing protein analysis services since 1987.
  • Partnered with Dr. Costel Darie's MS Lab at Clarkson University since 2013
  • FDA registered standardized method and training SOPs are followed with GMP analysis available.
  • Experience with a variety of samples including animal tissues, cultured cells, and purified protein products.

    • Detection Methods

    Coomassie BlueSilverWestern Blot
    SensitivityGood (~20 ng)Better (~2 ng)Best (pg to fg)
    QuantitativeYesSemi-quantitativeYes
    Best ForProtein quantitationLow abundance proteinsTrace protein detection

     

    References

    1. Korchak, J.A., et al., Proteoform medicine: characterizing and targeting protein forms in human disease. Nat Rev Genet, 2026.
    2. Su, J., et al., Personalized Drug Therapy: Innovative Concept Guided With Proteoformics. Mol Cell Proteomics, 2024. 23(3): p. 100737.
    3. Li, J. and X. Zhan, Mass spectrometry analysis of phosphotyrosine-containing proteins. Mass Spectrom Rev, 2024. 43(4): p. 857-887.
    4. Kendrick, N., et al., 2D SDS PAGE in Combination with Western Blotting and Mass Spectrometry Is a Robust Method for Protein Analysis with Many Applications. Adv Exp Med Biol, 2019. 1140: p. 563-574.
    5. Kendrick, N.D., C; Hoelter, M; Koll, A.; and Johansen, J. , Identification of Phosphotyrosine-Mutant Desmin in Human Pancreatic Cancer. BioRxiv, 2024.