Systematical optimization of reverse-phase chromatography for shotgun proteomics.
Systematical optimization of reverse-phase chromatography for shotgun proteomics.
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DOI：
10.1021/pr900251d
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发表时间：
2009-08
影响因子：
4.4
通讯作者：
Peng, Junmin
中科院分区：
文献类型：
--
作者：
Xu, Ping;Duong, Duc M.;Peng, Junmin
关键词：
我们报道了一个通用的LC/MS/MS平台的优化，以最大限度地从复杂的生物样本中识别出蛋白质的数量。该平台使用消化的酵母裂解物，在75μm内径×12 cm的反相柱上与LTQ-Orbitrap质谱仪相结合。我们首先产生了一种酵母肽混合物，并通过多种方法进行了定量，其中包括细胞培养中氨基酸稳定同位素标记策略(SILAC)。在高重复性、自动化的NanoLC/MS/MS系统上对多肽混合物进行分析，系统地调整上样量、流速、洗脱梯度范围和长度。有趣的是，通过加载~1μg的样品，柱几乎饱和。而最佳流速(~0.2min L/μ)和洗脱缓冲液范围(13~32%乙腈)与上样量无关，最佳梯度长度因进样量不同而不同：1μg多肽混合物160min，10 ng多肽40min。考察了这些参数对洗脱肽峰宽的影响。经过充分的优化，在一次160min的LC/MS/MS运行中，从1μg酵母裂解物中鉴定出1,012个蛋白质(聚集在806个组)，估计蛋白质假发现率为3%。
We report the optimization of a common LC/MS/MS platform to maximize the number of proteins identified from a complex biological sample. The platform uses digested yeast lysate on a 75 μm internal diameter × 12 cm reverse-phase column that is combined with an LTQ-Orbitrap mass spectrometer. We first generated a yeast peptide mix that was quantified by multiple methods including the strategy of stable isotope labeling with amino acids in cell culture (SILAC). The peptide mix was analyzed on a highly reproducible, automated nanoLC/MS/MS system with systematic adjustment of loading amount, flow rate, elution gradient range and length. Interestingly, the column was found to be almost saturated by loading ~1 μg of the sample. Whereas the optimal flow rate (~0.2 μl/min) and elution buffer range (13–32% of acetonitrile) appeared to be independent of the loading amount, the best gradient length varied according to the amount of samples: 160 min for 1 μg of the peptide mix, but 40 min for 10 ng of the same sample. The effect of these parameters on elution peptide peak width is evaluated. After full optimization, 1,012 proteins (clustered in 806 groups) with an estimated protein false discovery rate of ~3% were identified in 1 μg of yeast lysate in a single 160-min LC/MS/MS run.
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DOI：
10.1016/s1044-0305(02)00384-7
发表时间：
2002-06-01
影响因子：
3.2
作者：
Schwartz, JC;Senko, MW;Syka, JEP
通讯作者：
Syka, JEP
影响因子：
7.4
作者：
MacNair, JE;Lewis, KC;Jorgenson, JW
通讯作者：
Jorgenson, JW
影响因子：
4.4
作者：
Peng, JM;Elias, JE;Gygi, SP
通讯作者：
Gygi, SP
影响因子：
7.4
作者：
Seyfried, Nicholas T.;Xu, Ping;Peng, Junmin
通讯作者：
Peng, Junmin
影响因子：
64.5
作者：
Spence, J;Gali, RR;Finley, D
通讯作者：
Finley, D