QUANTITATIVE DEAMIDATION OF PEPTIDES

点击翻译按钮获取中文摘要

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Deamidation of proteins is a spontaneous, non-enzymatic post translational modification of proteins. It affects essentially all proteins, all the time, and as such represents the most common of all post translational modifications in proteins. The fact that deamidation occurs with such predictable regularity means that these modifications can be interpreted as molecular clocks [Robinson, 2001;Robinson, 2001;Robinson, 2001] with deamidation of proteins and the resulting unfolding of the protein being a putative signal for trafficking of the protein into the degradation machinery of the cell. However, this time-controlled signal is also implicated in many disease states, from amyloid to diabetes to autoimmune diseases. The full implications of deamidation are not known, and they are largely understudied because of the difficulty of a) determining deamidation extent accurately, and b) determining the ratios of the products. The first, determining the extent of deamidation, is difficult because the heavy isotope peaks of the Asn species overlap with the monoisotopic peak and isotopes of the Asp/isoAsp products. However, this mode can be studied fairly accurately by low resolution mass spectrometry if good isotopic ratios of the precursors are known by fitting the measured isotope ratios to a sum of individual isotopic distributions. Furthermore, if high resolution data is available, the 13C isotope peaks from Asn and the 12C isotopic peaks from Asp/isoAsp can be separated as they generally have a ~10 mDa difference in mass defect. The second problem, however, is much more difficult because it involves differentiating isomeric species of aspartic acid. They have the same mass and almost the same chemical reactivity in all circumstances. Very high resolution HPLC can sometimes be used to separate them (on a peptide by peptide basis), but even then its not always clear which peak is the Asp and which is the isoAsp. Furthermore, for detection in HPLC, its usually necessary to have milligram quantities of the peptide.