State Key Laboratory for Quality Research of Chinese Medicines, Macau University of Science and Technology , Macao, China.
Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical Garden, Chinese Academy of Sciences, Sino-Africa Joint Research Center, Chinese Academy of Sciences , Wuhan, China.
J Proteome Res. 2017 Sep 1;16(9):3470-3475. doi: 10.1021/acs.jproteome.7b00443. Epub 2017 Aug 17.
The usage of strong cation exchange (SCX) chromatography in proteomics is limited by its poor resolution and nonspecific hydrophobic interactions with peptides, which lead to peptide overlap across fractions and change of peptide retention, respectively. The application of high concentration of salt (up to 1000 mM) in SCX also restricted its use in online 2D SCX-RP LC. In the present research, we first exploited the chromatographic ability of online 2D SCX-RP LC by combination of acid, salt, and pH gradient, three relatively independent modes of eluting peptides from SCX column. 50% ACN was added to elution buffer for eliminating hydrophobic interactions between SCX matrix and peptides, and the concentration of volatile salt was reduced to 50 mM. Acid/salt/pH gradient showed superior resolution and sensitivity as well as uniform distribution across fractions, consequently leading to significant improvements in peptide and protein identification. 112 191 unique peptides and 7373 proteins were identified by acid/salt/pH fractionation, while 69 870 unique peptides and 4536 proteins were identified by salt elution, that is, 62.5 and 60.6% more proteins and unique peptides, respectively, identified by the former. Fraction overlap was also significantly minimized by acid/salt/pH approach. Furthermore, acid/salt/pH elution showed more identification for acidic peptides and hydrophilic peptides.
在蛋白质组学中，强阳离子交换（SCX）色谱的应用受到其分辨率差和与肽的非特异性疏水相互作用的限制，这分别导致肽在馏分之间重叠和肽保留时间发生变化。SCX 中高浓度盐（高达 1000mM）的应用也限制了其在在线二维 SCX-RP LC 中的应用。在本研究中，我们首先通过组合酸、盐和 pH 梯度三种相对独立的洗脱模式，利用在线二维 SCX-RP LC 的色谱能力来洗脱 SCX 柱上的肽。在洗脱缓冲液中加入 50%乙腈以消除 SCX 基质与肽之间的疏水相互作用，并将挥发性盐的浓度降低至 50mM。酸/盐/pH 梯度显示出优越的分辨率和灵敏度以及馏分之间均匀的分布，从而显著提高了肽和蛋白质的鉴定。通过酸/盐/pH 分级可鉴定出 112191 个独特肽和 7373 个蛋白质，而通过盐洗脱可鉴定出 69870 个独特肽和 4536 个蛋白质，即前者分别鉴定出 62.5%和 60.6%更多的蛋白质和独特肽。酸/盐/pH 方法还显著减少了馏分重叠。此外，酸/盐/pH 洗脱对酸性肽和亲水性肽的鉴定也更多。
