Flensburg John, Tangen Anders, Prieto Maria, Hellman Ulf, Wadensten Henrik
Amersham Biosciences AB, GE Healthcare, Björkgatan 30, SE-751 84 Uppsala, Sweden.
Eur J Mass Spectrom (Chichester). 2005;11(2):169-79. doi: 10.1255/ejms.734.
Derivatization of tryptic peptides using an Ettan CAF matrix-assisted laser desorption/ionization (MALDI) sequencing kit in combination with MALDI-post source decay (PSD) is a fast, accurate and convenient way to obtain de novo or confirmative peptide sequencing data. CAF (chemically assisted fragmentation) is based on solid-phase derivatization using a new class of water stable sulfonation agents, which strongly improves PSD analysis and simplifies the interpretation of acquired spectra. The derivatization is performed on solid supports, ZipTip(microC18, limiting the maximum peptide amount to 5 microg. By performing the derivatization in solution enabled the labeling of tryptic peptides derived from 100 microg of protein. To increase the number of peptides that could be sequenced, derivatized peptides were purified using multidimensional liquid chromatography (MDLC) prior to MALDI sequencing. Following the first dimension strong cation exchange (SCX) chromatography step, modified peptides were separated using reversed-phase chromatography (RPC). During the SCX clean up step, positively charged peptides are retained on the column while properly CAF-derivatized peptides (uncharged) are not. A moderately complex tryptic digest, prepared from six different proteins of equimolar amounts, was CAF-derivatized and purified by MDLC. Fractions from the second dimension nano RPC step were automatically sampled and on-line dispensed to MALDI sample plates and analyzed using MALDI mass spectrometry fragmentation techniques. All proteins in the derivatized protein mixture digest were readily identified using MALDI-PSD or MALDI tandem mass spectrometry (MS/MS). More than 40 peptides were unambiguously sequenced, representing a seven-fold increase in the number of sequenced peptides in comparison to when the CAF-derivatized protein mix digest was analyzed directly (no MDLC-separation) using MALDI-PSD. In conclusion, MDLC purification of CAF-derivatized peptides significantly increases the success rate for de novo and confirmative sequencing using various MALDI fragmentation techniques. This new approach is not only applicable to single protein digests but also to more complex digests and could, thus, be an alternative to electrospray ionization MS/MS for peptide sequencing.
使用Ettan CAF基质辅助激光解吸/电离（MALDI）测序试剂盒结合MALDI源后衰变（PSD）对胰蛋白酶肽进行衍生化，是一种快速、准确且便捷的方法，可用于获得从头测序或确证性的肽测序数据。CAF（化学辅助碎裂）基于使用一类新型水稳定磺化剂的固相衍生化，这极大地改善了PSD分析并简化了对所获光谱的解读。衍生化在固相载体ZipTip（microC18）上进行，将最大肽量限制为5微克。通过在溶液中进行衍生化，能够对源自100微克蛋白质的胰蛋白酶肽进行标记。为了增加可测序肽的数量，在进行MALDI测序之前，使用多维液相色谱（MDLC）对衍生化肽进行纯化。在第一维强阳离子交换（SCX）色谱步骤之后，使用反相色谱（RPC）分离修饰后的肽。在SCX净化步骤中，带正电荷的肽保留在柱上，而经过适当CAF衍生化的肽（不带电荷）则不会。用等摩尔量的六种不同蛋白质制备了中等复杂度的胰蛋白酶消化物，对其进行CAF衍生化并通过MDLC纯化。来自第二维纳米RPC步骤的馏分被自动取样并在线分配到MALDI样品板上，并使用MALDI质谱碎裂技术进行分析。使用MALDI-PSD或MALDI串联质谱（MS/MS）能够轻松鉴定衍生化蛋白质混合物消化物中的所有蛋白质。超过40个肽被明确测序，与直接使用MALDI-PSD分析CAF衍生化蛋白质混合消化物（未进行MDLC分离）相比，测序肽的数量增加了七倍。总之，对CAF衍生化肽进行MDLC纯化显著提高了使用各种MALDI碎裂技术进行从头测序和确证性测序的成功率。这种新方法不仅适用于单一蛋白质消化物，也适用于更复杂的消化物，因此，可作为肽测序的电喷雾电离MS/MS的替代方法。
