Optimizing performance of glycopeptide capture for plasma proteomics.
Optimizing performance of glycopeptide capture for plasma proteomics.
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DOI：
10.1021/pr900845m
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发表时间：
2010-04-05
影响因子：
4.4
通讯作者：
Carr, Steven A.
中科院分区：
文献类型：
--
作者：
Berven, Frode S.;Ahmad, Rushdy;Clouser, Karl R.;Carr, Steven A.
关键词：
选择性捕获糖共肽，然后释放和分析前糖基化位点肽，已被证明有希望降低体液（如血液）的复杂性，以发现生物标志物。在这项工作中，利用市售磁珠与肼化学结合，从人血浆中捕获含有n -连接碳水化合物的多肽的方案得到了优化，并通过使用KingFisher磁颗粒处理器实现了部分自动化。蛋白水平和肽水平的糖捕获比较揭示了检测到的前糖基化位点肽的特异性、可重复性和绝对数量的差异。通过对一系列捕获和洗脱条件的评估，得出了优化的方案，日内CV为24%，日间CV为30%，糖肽捕获特异性为99%。与未耗尽血浆相比，血浆中14种高丰度蛋白质的消耗提高了检测灵敏度约一个数量级，并导致鉴定的糖蛋白数量增加24%。SPEG检测无分离血浆中糖蛋白的灵敏度在10到100 pmol/ml范围内，对应于糖蛋白水平在100纳克/ml到10微克/ml之间。尽管具有很高的捕获特异性，但血浆中检测到的糖蛋白总数和SPEG的敏感性却令人惊讶地有限。
Selective capture of glycopolypeptides followed by release and analysis of the former glycosylation-site peptides has been shown to have promise for reducing complexity of body fluids such as blood for biomarker discovery. In this work, a protocol based on capture of polypeptides containing a N-linked carbohydrate from human plasma using commercially available magnetic beads coupled with hydrazide chemistry was optimized and partially automated through the use of a KingFisher magnetic particle processor. Comparison of bead-based glycocapture at the protein-level vs. peptide-level revealed differences in the specificity, reproducibility and absolute number of former glycosylation-site peptides detected. Evaluation of a range of capture and elution conditions led to an optimized protocol with a 24% intraday and 30% interday CV, and a glycopeptide capture specificity of 99%. Depleting the plasma of 14 high abundance proteins improved detection sensitivity by approximately one order of magnitude compared to non-depleted plasma and resulted in an increase of 24% in the number of identified glycoproteins. The sensitivity of SPEG for detection of glycoproteins in depleted, non-fractionated plasma was found to be in the 10 to 100 pmol/ml range corresponding to glycoprotein levels ranging from 100’s of nanograms/ml to 10’s of micrograms/ml. Despite high capture specificity,the total number of glycoproteins detected and the sensitivity of SPEG in plasma is surprisingly limited.
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影响因子：
3.1
作者：
Rajcevic, Uros;Niclou, Simone P.;Jimenez, Connie R.
通讯作者：
Jimenez, Connie R.
DOI：
10.1016/0021-9673(92)80101-y
发表时间：
1992-04-24
期刊：
JOURNAL OF CHROMATOGRAPHY
影响因子：
--
作者：
KOBATA, A;ENDO, T
通讯作者：
ENDO, T
影响因子：
2.7
作者：
Schenk, Susann;Schoenhals, Gary J.;de Souza, Gustavo;Mann, Matthias
通讯作者：
Mann, Matthias
影响因子：
7.4
作者：
Zhou, Yong;Aebersold, Ruedi;Zhang, Hui
通讯作者：
Zhang, Hui
影响因子：
4.4
作者：
Liu, T;Qian, WJ;Smith, RD
通讯作者：
Smith, RD