Mapping preferred sites for fluorescent labeling by combining fluorescence and MS analysis of tryptic CNA35 protein digests.
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.
Mingels
Alma M A
AM
Laboratory of Macromolecular and Organic Chemistry, Department of Biomedical Engineering, Eindhoven University of Technology, PO Box 513, 5600 MB Eindhoven, The Netherlands.
van Dongen
Joost L J
JL
Merkx
Maarten
M
D018829:Adhesins, Bacterial / Q000032:analysis* / Q000378:metabolism; D000818:Animals; D002264:Carboxylic Acids / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003094:Collagen / Q000032:analysis / Q000378:metabolism; D005456:Fluorescent Dyes / Q000032:analysis* / Q000378:metabolism; D008239:Lysine / Q000032:analysis / Q000737:chemistry; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010449:Peptide Mapping; D011485:Protein Binding; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013194:Staining and Labeling; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014357:Trypsin / Q000378:metabolism
D018829:Adhesins, Bacterial; D002264:Carboxylic Acids; D005456:Fluorescent Dyes; C436864:Oregon Green 488 carboxylic acid; D003094:Collagen; D014357:Trypsin; D008239:Lysine