Strickler J E, Hunkapiller M W, Wilson K J
Anal Biochem. 1984 Aug 1;140(2):553-66. doi: 10.1016/0003-2697(84)90207-0.
The utility of the commercially available gas-phase sequencer for complete analysis of peptide samples was investigated. Using the program supplied with the instrument, significant extractive loss of samples in Polybrene was observed, even at input levels up to 500 pmol. In order to reduce this loss, the sequencer program was modified by increasing the phenylisothiocyanate (PITC)-coupling steps from two to three and lengthening the duration of ethyl acetate (S2) delivery while reducing the delivery rate. These changes gave improved results with peptides, e.g., all eight residues of angiotensin II were identified at the 25-pmol level. In addition, background contamination was decreased and repetitive yields were increased. The instrument was also found to function well with samples coupled to solid supports; however, some of the methodologies that work adequately for covalent attachment of peptides to solid supports at the level 1-10 nmol were found to give unacceptable coupling/sequenceable yields at or below the 100-pmol level. The coupling methods tried were (1) reaction of homoserine lactone with aminopropyl (AP)-glass, (2) reaction of alpha- and epsilon-NH2 groups with p-phenylenediisothiocyanate (DITC)-glass, and (3) reaction of alpha-COOH groups with aminoaryl (AA)-glass via EDAC (1-ethyl-3,3'-dimethylaminopropyl-carbodiimide). Of these, the first method gave combined yields of 42-94% while the latter two were only 9-35% efficient. The covalently bound samples provided sequence information even at the resulting low levels, e.g., 9/13 residues of dynorphin including Lys-13 at 11 pmol. In general, sequencer runs on solid-phase samples gave "cleaner" analyses and slightly higher repetitive yields (1-2%). Sequence information has also been obtained on peptides made by solid-phase synthesis prior to cleavage from the polystyrene support. With improved coupling efficiencies, solid-phase techniques would provide an alternative to immobilization of peptides in Polybrene films for low picomole level gas-phase sequencing.
对市售气相测序仪用于肽样品完整分析的实用性进行了研究。使用仪器附带的程序，即使在输入水平高达500皮摩尔时，也观察到聚凝胺中样品有显著的提取损失。为了减少这种损失，对测序仪程序进行了修改，将异硫氰酸苯酯（PITC）偶联步骤从两步增加到三步，并延长乙酸乙酯（S2）输送的持续时间，同时降低输送速率。这些改变对肽产生了更好的结果，例如，在25皮摩尔水平上鉴定出了血管紧张素II的所有八个残基。此外，背景污染减少，重复产率提高。还发现该仪器对与固相支持物偶联的样品也能很好地发挥作用；然而，一些在1 - 10纳摩尔水平上能充分用于肽与固相支持物共价连接的方法，发现在100皮摩尔及以下水平时，偶联/可测序产率不可接受。尝试的偶联方法有：（1）高丝氨酸内酯与氨丙基（AP）玻璃的反应；（2）α-氨基和ε-氨基与对苯二异硫氰酸酯（DITC）玻璃的反应；（3）α-羧基通过1-乙基-3,3'-二甲基氨基丙基碳二亚胺（EDAC）与氨基芳基（AA）玻璃的反应。其中，第一种方法的综合产率为42 - 94%，而后两种方法的效率仅为9 - 35%。即使在所得的低水平下，共价结合的样品也能提供序列信息，例如，在11皮摩尔时，强啡肽的13个残基中有9个被鉴定出来，包括第13位的赖氨酸。一般来说，对固相样品进行测序仪运行能得到“更清晰”的分析结果，且重复产率略高（1 - 2%）。在从聚苯乙烯支持物上切割下来之前，也已经获得了通过固相合成制备的肽的序列信息。随着偶联效率的提高，固相技术将为在低皮摩尔水平气相测序中，将肽固定在聚凝胺膜中提供一种替代方法。
