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Chemo-Selection Strategy for Limited Proteolysis Experiments on the Proteomic Scale.

Publication ,  Journal Article
Ma, R; Meng, H; Wiebelhaus, N; Fitzgerald, MC
Published in: Analytical chemistry
December 2018

Described here is a chemo-selective enrichment strategy, termed the semitryptic peptide enrichment strategy for proteolysis procedures (STEPP), to isolate the semitryptic peptides generated in mass spectrometry-based proteome-wide applications of limited proteolysis methods. The strategy involves reacting the ε-amino groups of lysine side chains and any N-termini created in the limited proteolysis reaction with isobaric mass tags. A subsequent digestion of the sample with trypsin and the chemo-selective reaction of the newly exposed N-termini of the tryptic peptides with N-hydroxysuccinimide (NHS)-activated agarose resin removes the tryptic peptides from solution, leaving only the semitryptic peptides with one nontryptic cleavage site generated in the limited proteolysis reaction for subsequent LC-MS/MS analysis. As part of this work, the STEPP technique is interfaced with two different proteolysis methods, including the pulse proteolysis (PP) and limited proteolysis (LiP) methods. The STEPP-PP workflow is evaluated in two proof-of-principle experiments involving the proteins in a yeast cell lysate and two well-studied drugs, cyclosporin A and geldanamycin. The STEPP-LiP workflow is evaluated in a proof-of-principle experiment involving the proteins in two cell culture models of human breast cancer, MCF-7 and MCF-10A cell lines. The STEPP protocol increased the number of semitryptic peptides detected in the LiP and PP experiments by 5- to 10-fold. The STEPP protocol not only increases the proteomic coverage, but also increases the amount of structural information that can be gleaned from limited proteolysis experiments. Moreover, the protocol also enables the quantitative determination of ligand binding affinities.

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Published In

Analytical chemistry

DOI

EISSN

1520-6882

ISSN

0003-2700

Publication Date

December 2018

Volume

90

Issue

23

Start / End Page

14039 / 14047

Related Subject Headings

  • Tumor Cells, Cultured
  • Tandem Mass Spectrometry
  • Proteomics
  • Proteolysis
  • Peptides
  • MCF-7 Cells
  • Humans
  • Chromatography, Liquid
  • Analytical Chemistry
  • 4004 Chemical engineering
 

Citation

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Ma, R., Meng, H., Wiebelhaus, N., & Fitzgerald, M. C. (2018). Chemo-Selection Strategy for Limited Proteolysis Experiments on the Proteomic Scale. Analytical Chemistry, 90(23), 14039–14047. https://doi.org/10.1021/acs.analchem.8b04122
Ma, Renze, He Meng, Nancy Wiebelhaus, and Michael C. Fitzgerald. “Chemo-Selection Strategy for Limited Proteolysis Experiments on the Proteomic Scale.Analytical Chemistry 90, no. 23 (December 2018): 14039–47. https://doi.org/10.1021/acs.analchem.8b04122.
Ma R, Meng H, Wiebelhaus N, Fitzgerald MC. Chemo-Selection Strategy for Limited Proteolysis Experiments on the Proteomic Scale. Analytical chemistry. 2018 Dec;90(23):14039–47.
Ma, Renze, et al. “Chemo-Selection Strategy for Limited Proteolysis Experiments on the Proteomic Scale.Analytical Chemistry, vol. 90, no. 23, Dec. 2018, pp. 14039–47. Epmc, doi:10.1021/acs.analchem.8b04122.
Ma R, Meng H, Wiebelhaus N, Fitzgerald MC. Chemo-Selection Strategy for Limited Proteolysis Experiments on the Proteomic Scale. Analytical chemistry. 2018 Dec;90(23):14039–14047.
Journal cover image

Published In

Analytical chemistry

DOI

EISSN

1520-6882

ISSN

0003-2700

Publication Date

December 2018

Volume

90

Issue

23

Start / End Page

14039 / 14047

Related Subject Headings

  • Tumor Cells, Cultured
  • Tandem Mass Spectrometry
  • Proteomics
  • Proteolysis
  • Peptides
  • MCF-7 Cells
  • Humans
  • Chromatography, Liquid
  • Analytical Chemistry
  • 4004 Chemical engineering