Gheorghe M T, Jörnvall H, Bergman T
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, S-171 77, Sweden.
Anal Biochem. 1997 Dec 1;254(1):119-25. doi: 10.1006/abio.1997.2380.
N-terminal protein acetylation is a common posttranslational modification, blocking Edman degradation during sequencer analysis. Use of mass spectrometry allows the analysis also of acetyl-blocked polypeptides; however, for large proteins mass spectrometry is not always informative, and deacetylation by chemical pretreatments is desirable for making direct sequencer analysis possible. For this purpose, alcoholytic deacetylation is attractive. In the present work, we have studied the optimal conditions for specific removal of the acetyl group without extensive cleavage of peptide bonds in general. We find that incubation with trifluoroacetic acid in methanol (1:1, by volume) at an elevated temperature ( approximately 47 degrees C) for 2-3 days results in efficient deacetylation allowing direct application to sequencer analysis with initial yields up to approximately 50% of the amount applied for deblocking. Deacetylation compared to internal peptide bond cleavage is often high, as evaluated by recoveries of residues from the deblocked sequence over those from the background, and this applies to both peptides (up to the order of 10:1 for the specific residue versus the background) and proteins (>2:1). Although yields may still vary and some sequences be only partly susceptible to the chemistry, this deblocking can in many cases allow unambiguous interpretation of N-terminally acetyl-blocked sequences.
N 端蛋白质乙酰化是一种常见的翻译后修饰，会在序列分析过程中阻碍埃德曼降解。质谱分析的应用使得对乙酰化阻断的多肽也能进行分析；然而，对于大型蛋白质，质谱分析并不总是能提供足够信息，因此通过化学预处理进行脱乙酰化对于实现直接序列分析是很有必要的。为此，醇解脱乙酰化很有吸引力。在本研究中，我们总体上研究了在不大量裂解肽键的情况下特异性去除乙酰基的最佳条件。我们发现，在高温（约47℃）下，将其与甲醇（体积比1:1）中的三氟乙酸孵育2 - 3天，可实现高效脱乙酰化，从而能够直接用于序列分析，初始产率可达用于去封闭的量的约50%。通过去封闭序列中残基的回收率与背景中残基的回收率进行评估，与内部肽键裂解相比，脱乙酰化效率通常较高，这适用于肽（特定残基与背景的回收率高达10:1）和蛋白质（>2:1）。尽管产率可能仍会有所不同，且某些序列可能仅部分易受该化学方法影响，但这种去封闭在许多情况下能够明确解读N端乙酰化阻断的序列。
