Apffel A, Fischer S, Goldberg G, Goodley P C, Kuhlmann F E
Bay Analytical Operation, Hewlett-Packard Co., Palo Alto, CA 94304, USA.
J Chromatogr A. 1995 Sep 29;712(1):177-90. doi: 10.1016/0021-9673(95)00175-m.
A method is described for improving the sensitivity of peptide mapping with electrospray liquid chromatography--mass spectrometry using trifluoroacetic acid (TFA) containing HPLC mobile phases. The signal suppressing effects of TFA are shown to be due to the combined effect of ion-pairing and surface tension modifications. The post-column addition of a propionic acid-2-propanol (75:25, v/v) in a 1:2 proportion with the HPLC mobile phase counteracts the deleterious effects of TFA resulting in 10-100 x improvement of the signal-to-noise ratio. The system described introduces total HPLC flow (plus additive) directly into the electrospray source without splitting. Using 2.1 mm I.D. HPLC columns, minimum detectable quantities are below 40 pmol total protein. As examples, separations of proteolytic enzyme digests of several proteins are shown using standard HPLC conditions, comparing results with and without the addition of propionic acid. The application of the technique is shown in more depth in the identification of oxidative modification sites in glutamine synthetase. In this application, the enhanced sensitivity allowed location of a modified residue by comparison endoproteinase Lys C digest of native and oxidized forms of the protein without extensive sample preparation or concentration. A third application demonstrates the identification of glycosylation sites in an endoproteinase Arg C digest of single-chain plasminogen activator through the use of in-source collisionally induced dissociation.
本文描述了一种使用含三氟乙酸（TFA）的高效液相色谱流动相，通过电喷雾液相色谱 - 质谱联用提高肽图谱分析灵敏度的方法。结果表明，TFA的信号抑制作用是离子对作用和表面张力改变共同作用的结果。在柱后以1:2的比例添加丙酸 - 2 - 丙醇（75:25，v/v）与高效液相色谱流动相，可抵消TFA的有害影响，使信噪比提高10 - 100倍。所描述的系统将高效液相色谱总流量（加添加剂）直接引入电喷雾源而不进行分流。使用内径为2.1 mm的高效液相色谱柱，总蛋白的最低检测量低于40 pmol。作为示例，展示了在标准高效液相色谱条件下对几种蛋白质的蛋白酶消化产物的分离，比较了添加和不添加丙酸的结果。该技术在谷氨酰胺合成酶氧化修饰位点鉴定中的应用更深入地展示了这一点。在此应用中，增强的灵敏度使得通过比较蛋白质天然形式和氧化形式的内肽酶Lys C消化产物，无需大量样品制备或浓缩即可定位修饰残基。第三个应用展示了通过源内碰撞诱导解离鉴定单链纤溶酶原激活剂的内肽酶Arg C消化产物中的糖基化位点。
