Top-down characterization of proteins and drug-protein complexes using nanoelectrospray tandem mass spectrometry.
We report a 'top-down' approach for characterization of proteins, and identification of binding sites in protein-drug complexes using nanoelectrospray ionization hybrid quadrupole time-of-flight tandem mass spectrometry (nanoESI-MS/MS). The efficiency of direct fragmentation of intact protein ions and the feasibility of this method were initially demonstrated using several well-characterized proteins with different molecular weights including metallothionein (6126 Da), cytochrome c (horse, 12360 Da), myoglobin (horse, 16592 Da), and hemoglobin (human, 64453 Da). Simply varying collision energy without enzyme digestion and gel or LC separation generated a range of peptide fragments of these proteins. Over 80% of these peptide ions matched those in the SWISS-PROT database with mass accuracy of 8 to 32 ppm with external calibration. This technique was further applied to fragment a cisplatin-metallothionein complex to identify the binding sites, demonstrating a potential application in the study of drug-protein binding.
Mandal
Rupasri
R
Environmental Health Sciences, Department of Public Health Sciences, Faculty of Medicine and Dentistry, University of Alberta, 10-102 Clinical Sciences Building, Edmonton, AB Canada.
Li
Xing-Fang
XF
D000595:Amino Acid Sequence; D000818:Animals; D000970:Antineoplastic Agents / Q000378:metabolism; D001665:Binding Sites; D002945:Cisplatin / Q000737:chemistry* / Q000378:metabolism; D045304:Cytochromes c / Q000737:chemistry* / Q000378:metabolism; D006736:Horses; D008668:Metallothionein / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D009211:Myoglobin / Q000737:chemistry* / Q000378:metabolism; D036103:Nanotechnology / Q000379:methods*; D011485:Protein Binding; D011817:Rabbits; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D000970:Antineoplastic Agents; D009211:Myoglobin; D045304:Cytochromes c; D008668:Metallothionein; D002945:Cisplatin