QUANTITATIVE DEAMIDATION OF PEPTIDES

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 蛋白质的脱酰胺是蛋白质的自发的、非酶促的翻译后修饰。 它基本上影响所有的蛋白质，所有的时间，因此代表了最常见的所有蛋白质翻译后修饰。 脱酰胺以这种可预测的规律性发生的事实意味着这些修饰可以被解释为分子钟[罗宾逊，2001;罗宾逊，2001;罗宾逊，2001]，其中蛋白质脱酰胺和由此产生的蛋白质解折叠是蛋白质运输到细胞降解机制中的推定信号。 然而，这种时间控制的信号也与许多疾病状态有关，从淀粉样蛋白到糖尿病再到自身免疫性疾病。 脱酰胺的全部含义尚不清楚，并且由于a）准确测定脱酰胺程度和B）测定产物比率的困难，它们在很大程度上未得到充分研究。 第一，确定脱酰胺化的程度，是困难的，因为Asn物质的重同位素峰与Asp/isoAsp产物的单一同位素峰和同位素重叠。 然而，这种模式可以相当准确地研究低分辨率质谱法，如果良好的同位素比的前体是已知的，通过拟合测量的同位素比的个别同位素分布的总和。 此外，如果可获得高分辨率数据，则可以分离来自Asn的13 C同位素峰和来自Asp/isoAsp的12 C同位素峰，因为它们的质量亏损通常相差约10 mDa。 然而，第二个问题要困难得多，因为它涉及到区分天冬氨酸的异构体。 它们在任何情况下都具有相同的质量和几乎相同的化学反应性。 有时可以使用非常高分辨率的HPLC来分离它们（基于逐个肽），但即使这样，并不总是清楚哪个峰是Asp，哪个峰是isoAsp。 此外，对于HPLC检测，通常需要毫克量的肽。

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.