QUANTITATIVE DEAMIDATION OF PEPTIDES

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 蛋白质去酰胺化是一种自发的、非酶的蛋白质翻译后修饰。它基本上一直影响着所有的蛋白质，因此代表了蛋白质中最常见的翻译后修饰。脱酰胺化的发生具有如此可预测的规律性，这意味着这些修饰可以被解释为分子时钟[Robinson，2001；Robinson，2001；Robinson，2001]，其中蛋白质的脱酰胺化和由此产生的蛋白质的展开是蛋白质运输到细胞降解机制的假定信号。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。脱酰胺化的全部影响尚不清楚，而且由于a)准确确定脱酰胺化程度和b)确定产物比例的困难，对它们的研究在很大程度上是不够的。 第一，确定脱酰胺的程度是困难的，因为ASN物种的重同位素峰与Asp/isAsp产物的单同位素峰和同位素重叠。然而，如果通过将测量的同位素比与单个同位素分布的总和进行拟合，知道前驱体的良好同位素比，那么这种模式可以用低分辨率质谱学相当准确地研究。此外，如果有高分辨率数据，Asn的13C同位素峰和Asp/isAsp的12C同位素峰可以分离，因为它们的质量缺陷通常有~10mda的差异。 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。它们在所有情况下都具有相同的质量和几乎相同的化学反应活性。有时可以使用非常高分辨率的高效液相色谱法来分离它们(逐个多肽)，但即使这样，也不总是清楚哪个峰是天冬氨酸，哪个是等天冬氨酸。此外，为了在高效液相色谱中进行检测，通常需要有毫克量的多肽。

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.