Quantification of isotopically overlapping deamidated and 18o-labeled peptides using isotopic envelope mixture modeling.
Quantification of isotopically overlapping deamidated and 18o-labeled peptides using isotopic envelope mixture modeling.
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DOI：
10.1021/pr801054w
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发表时间：
2009-03
影响因子：
4.4
通讯作者：
David LL
中科院分区：
文献类型：
--
作者：
Dasari S;Wilmarth PA;Reddy AP;Robertson LJ;Nagalla SR;David LL
开发了一种稳健的肽定量方法，其中重叠的肽同位素分布与预测的肽同位素包膜混合物模型（IEMM）拟合。应用到两个困难的定量问题进行了演示。第一个是脱酰胺的定量，其中同位素峰的质量相差一个Da，第二个是18 O标记，其中同位素峰偏移2和4 Da。在这两种情况下，肽定量不能通过提取的离子色谱图的简单积分进行，因为质量偏移肽的同位素包络线通常不能分辨。为了检测脱酰胺定量的方法，将几种合成肽及其相应的脱酰胺形式以不同比例（1：0、1：2、2：1、4：1、10：1和20：1）混合，并使用IEMM方法进行分析，导致测得的脱酰胺百分比与已知的脱酰胺百分比之间具有高度相关性（R2=0.96）。然后将IEMM方法纳入大规模蛋白质组学实验中脱酰胺定量的工作流程中。采用二维液相色谱串联质谱法分析了一系列正常（3天、2年、35年和70年）和白内障（93年）人晶状体，并测定了几种γ S-晶状体蛋白肽（[N14-Q16]、N53、[Q63-Q70]和N143）的脱酰胺量。两种肽（N53和[Q63-Q70]）在正常透镜样本的水不溶性部分中具有更广泛的脱酰胺作用，并且在93岁的水不溶性白内障样本中N143处的脱酰胺作用更广泛。使用已知16 O与18 O比例（10：1、4：1、1：1、1：4和1：10）的标记BSA肽混合物，检查了18 O标记肽分析技术的实用性。该方法允许的16 O/18 O肽的比例在广泛的相对丰度的准确测量。
A robust peptide quantification method was developed where overlapping peptide isotopic distributions were fit with predicted peptide isotopic envelope mixture models (IEMM). Application to two difficult quantitative problems was demonstrated. The first was the quantification of deamidation, where masses of isotopic peaks differ by a single Da, and the second, 18O labeling, where the isotopic peaks are shifted 2 and 4 Da. In both cases, peptide quantification cannot be performed by simple integration of extracted ion chromatograms, because the isotopic envelopes of mass shifted peptides are normally not resolved. To test the methodology for quantification of deamidation, several synthetic peptides and their corresponding deamidated forms were mixed at various ratios (1:0, 1:2, 2:1, 4:1, 10:1 and 20:1) and analyzed using the IEMM method, resulting in a high correlation (R2=0.96) between measured and known percentages of deamidation. The IEMM method was then incorporated into a workflow for deamidation quantification in a large-scale proteomics experiment. A series of normal (3-day, 2-year, 35-year, and 70-year) and cataractous (93-year) human lenses were analyzed using two-dimensional liquid chromatography tandem mass spectrometry and deamidation quantities of several γS-crystallin peptides ([N14-Q16], N53, [Q63–Q70], and N143) were determined. Two peptides (N53 and [Q63–Q70]) had more extensive deamidation in the water-insoluble portions of normal lens samples, and deamidation at N143 was more extensive in the 93-year water-insoluble cataractous sample. The utility of the technique for analysis of 18O-labeled peptides was examined using mixtures of labeled BSA peptides in known 16O to 18O ratios (10:1, 4:1, 1:1, 1:4, and 1:10). The methodology allowed accurate measurements of ratios of 16O/18O peptides over the wide range of relative abundances.
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影响因子：
7
作者：
Ramos-Fernandez, Antonio;Lopez-Ferrer, Daniel;Vazquez, Jesús
通讯作者：
Vazquez, Jesús
影响因子：
7.4
作者：
Searle, BC;Dasari, S;Nagalla, SR
通讯作者：
Nagalla, SR
影响因子：
4.4
作者：
Dasari, Surendra;Wilmarth, Phillip A.;David, Larry L.
通讯作者：
David, Larry L.
DOI：
10.1016/1044-0305(95)00017-8
发表时间：
1995-04-01
影响因子：
3.2
作者：
SENKO, MW;BEU, SC;MCLAFFERTY, FW
通讯作者：
MCLAFFERTY, FW
影响因子：
4.4
作者：
Tabb, DL;McDonald, WH;Yates, JR
通讯作者：
Yates, JR