Characterization of Proteins and Their Modifications by 1-D and 2-D LC/ESI-TOF MS Analysis of Complex Mixtures of Intact Proteins.

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Scott J. Berger;Hongji Liu;Asish Chakraborty;Robert S. Plumb;Steven A. Cohen [Waters]
FAll ACS 2002; 224th ACS National Meeting; Boston August 18-22
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Protein; Yeast Ribosomal Protein Fraction
Symmetry300(™) C4 300 Å 3.5 µm Steel 2.1 mm 50 mm
Mass spectrometry of intact proteins provides comprehensive data relating to the state of modification, but intact mass data alone does not necessarily provide for direct identification of a protein or modified protein, nor does it localize modification sites. Our group has developed an approach using 1-D and 2-D LC/MS to fully characterize intact proteins, and complicated protein mixtures. In this approach, 1-D (reversed phase) or 2-D strong cation-exchange/reversed-phase separations are coupled to online ESI-TOF mass detection, while diverting the bulk of eluent to UV/fraction collection. The collected fractions are processed to confirm identifications and identify modifications. Using protein mixtures as complex as a yeast ribosomal protein fraction (¡«100 proteins), we have been able to independently evaluate chromatographic performance of each separation dimension, systematically examine co-translational and posttranslational protein processing, and identify putative deamidation events for several proteins in the mixture.

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