Identification of Intact Proteins in Mixtures by Alternated Capillary Liquid Chromatography Electrospray Ionization and LC ESI Infrared Multiphoton Dissociation Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

Citations Over TimeTop 10% of 1999 papers

Abstract

Here we propose a novel method for rapidly identifying proteins in complex mixtures. A list of candidate proteins (including provision for posttranslational modifications) is obtained by database searching, within a specified mass range about the accurately measured mass (e.g., +/- 0.1 Da at 10 kDa) of the intact protein, by capillary liquid chromatography electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (LC ESI FT-ICR MS). On alternate scans, LC ESI infrared multiphoton dissociation (IRMPD) FT-ICR MS yields mostly b and y fragment ions for each protein, from which the correct candidate is identified as the one with the highest "hit" score (i.e., most b and y fragments matching the candidate database protein amino acid sequence masses) and sequence "tag" score (based on a series of fragment sequences differing in mass by 1 or 2 amino acids). The method succeeds in uniquely identifying each of a mixture of five proteins treated as unknowns (melittin, ubiquitin, GroES, myoglobin, carbonic anhydrase II), from more than 1000 possible database candidates within a +/- 500 Da mass window. We are also able to identify posttranslational modifications of two of the proteins (mellitin and GroES). The method is simple, rapid, and definitive and is extendable to a mixture of affinity-selected proteins, to identify proteins with a common biological function.

Related Papers

• → Identification of Intact Proteins in Mixtures by Alternated Capillary Liquid Chromatography Electrospray Ionization and LC ESI Infrared Multiphoton Dissociation Fourier Transform Ion Cyclotron Resonance Mass Spectrometry(1999)99 cited
• → Differentiating Fragmentation Pathways of Cholesterol by Two-Dimensional Fourier Transform Ion Cyclotron Resonance Mass Spectrometry(2015)40 cited
• → Application of electrospray ionization with Fourier transform ion cyclotron resonance mass spectrometry for structural screening of core oligosaccharides from lipopolysaccharides of the bacteria Proteus(2005)19 cited
• → Protein identification by peptide mass fingerprinting and peptide sequence tagging with alternating scans of nano‐liquid chromatography/infrared multiphoton dissociation Fourier transform ion cyclotron resonance mass spectrometry(2003)9 cited
• Ion Structure and Energetics in the Gas Phase Characterized Using Fourier Transform Ion Cyclotron Resonance Mass Spectrometry(2014)