We recently reported the use of desorption electrospray ionization (DESI) as a novel interface to couple high-performance liquid chromatography (HPLC) with mass spectrometry (MS) (Chem. Commun. 2011, 47, 4171). One of the benefits of such an interface is that post-column derivatization of separated analytes can be integrated with ionization via a "reactive" DESI approach in which a derivatizing reagent is doped into the spray solvent. The reactive DESI interface allows analyte desorption/ionization from the end of the chromatographic column with prompt MS detection; a short time delay of ~20 ms was demonstrated. In this study, we extended this application by "supercharging" proteins following HPLC separation using a DESI spray solvent containing supercharging reagents, m-nitrobenzyl alcohol (m-NBA) or sulfolane. Proteins (insulin, ubiquitin, lysozyme and α-lactalbumin) eluted out of the LC column can be supercharged with the protein charge state distributions (CSDs) significantly increased (to higher charge), which would be advantageous for subsequent top-down MS analysis of proteins. Interestingly, supercharging combined with reactive DESI enhances tolerance towards trifluoroacetic acid (TFA), which is known to be a superior additive in the mobile phase for premium peptide/protein chromatographic separation but has severe signal suppression effects for conventional electrospray ionization (ESI). In comparison to electrosonic spray ionization (ESSI), a variant form of ESI, the sensitivity of protein analysis using LC/DESI-MS with the mobile phase containing TFA can be improved by up to 70-fold for lysozyme and α-lactalbumin by including m-NBA in the DESI spray solvent. Presumably, by reducing TFA dissociation in the droplet, supercharging agents lower trifluoroacetate anion concentrations and concomitantly reduce ion pairing to analyte cationic sites. The reduced ion pairing therefore decreases the TFA signal suppression effect. The supercharging capability and the reduction of TFA signal suppression suggest that LC/DESI-MS is a valuable method for protein analysis.

中文翻译：

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使用解吸电喷雾电离的液相色谱-质谱法进行蛋白质分析的信号和电荷增强

我们最近报道了使用解吸电喷雾电离 (DESI) 作为将高效液相色谱 (HPLC) 与质谱 (MS) 结合的新型界面 (Chem. Commun. 2011, 47, 4171)。这种接口的好处之一是分离分析物的柱后衍生化可以通过“反应性”DESI 方法与电离结合，其中衍生化试剂被掺杂到喷雾溶剂中。反应性 DESI 接口允许从色谱柱末端解吸/电离分析物，并进行快速 MS 检测；展示了约 20 毫秒的短时间延迟。在这项研究中，我们通过使用含有增压试剂、间硝基苯甲醇 (m-NBA) 或环丁砜的 DESI 喷雾溶剂在 HPLC 分离后“增压”蛋白质来扩展此应用。从 LC 色谱柱中洗脱的蛋白质（胰岛素、泛素、溶菌酶和 α-乳清蛋白）可以随着蛋白质电荷状态分布 (CSD) 显着增加（至更高电荷）而增压，这将有利于随后自上而下的 MS 分析蛋白质。有趣的是，增压结合反应性 DESI 增强了对三氟乙酸 (TFA) 的耐受性，众所周知，三氟乙酸是用于优质肽/蛋白质色谱分离的流动相中的优良添加剂，但对传统电喷雾电离 (ESI) 具有严重的信号抑制作用。与 ESI 的一种变体形式的电声喷雾电离 (ESSI) 相比，通过在 DESI 喷雾溶剂中包含 m-NBA，使用含 TFA 的流动相使用 LC/DESI-MS 分析蛋白质的灵敏度可提高多达 70 倍，用于溶菌酶和 α-乳清蛋白。据推测，通过减少液滴中的 TFA 解离，增压剂降低了三氟乙酸根阴离子浓度，并同时减少了与分析物阳离子位点的离子配对。因此，减少的离子对降低了 TFA 信号抑制效果。增压能力和 TFA 信号抑制的减少表明 LC/DESI-MS 是一种有价值的蛋白质分析方法。因此，减少的离子对降低了 TFA 信号抑制效果。增压能力和 TFA 信号抑制的减少表明 LC/DESI-MS 是一种有价值的蛋白质分析方法。因此，减少的离子对降低了 TFA 信号抑制效果。增压能力和 TFA 信号抑制的减少表明 LC/DESI-MS 是一种有价值的蛋白质分析方法。