Mapping preferred sites for fluorescent labeling by combining fluorescence and MS analysis of tryptic CNA35 protein digests

Alma M A Mingels; Joost L J van Dongen; Maarten Merkx

doi:10.1016/j.jchromb.2007.12.026

Mapping preferred sites for fluorescent labeling by combining fluorescence and MS analysis of tryptic CNA35 protein digests

J Chromatogr B Analyt Technol Biomed Life Sci. 2008 Mar 1;863(2):293-7. doi: 10.1016/j.jchromb.2007.12.026. Epub 2008 Jan 16.

Authors

Alma M A Mingels¹, Joost L J van Dongen, Maarten Merkx

Affiliation

¹ Laboratory of Macromolecular and Organic Chemistry, Department of Biomedical Engineering, Eindhoven University of Technology, PO Box 513, 5600 MB Eindhoven, The Netherlands.

PMID: 18272440
DOI: 10.1016/j.jchromb.2007.12.026

Abstract

HPLC-MS analysis of tryptic protein digests in combination with fluorescence detection is presented as a convenient and quantitative method to gain insight into the relative reactivity of lysine side chains. In this scheme (tandem) mass spectrometry was used for identification of the modified residue, whereas fluorescence detection allowed determination of their relative abundance. Our method identified 'labeling hot-spots' at two flexible parts of the collagen-binding protein CNA35, positions that were consistent with all available structural and biochemical data on the collagen-binding properties of CNA35.

MeSH terms

Adhesins, Bacterial / analysis*
Adhesins, Bacterial / metabolism
Animals
Carboxylic Acids / chemistry
Chromatography, High Pressure Liquid / methods
Collagen / analysis
Collagen / metabolism
Fluorescent Dyes / analysis*
Fluorescent Dyes / metabolism
Lysine / analysis
Lysine / chemistry
Mice
Molecular Sequence Data
Molecular Weight
Peptide Mapping*
Protein Binding
Spectrometry, Mass, Electrospray Ionization / methods*
Staining and Labeling
Tandem Mass Spectrometry / methods*
Trypsin / metabolism

Substances

Adhesins, Bacterial
Carboxylic Acids
Fluorescent Dyes
Oregon Green 488 carboxylic acid
Collagen
Trypsin
Lysine