Zhang Z, Smith D L
Department of Medicinal Chemistry and Pharmacognosy, Purdue University, West Lafayette, Indiana 47907.
Protein Sci. 1993 Apr;2(4):522-31. doi: 10.1002/pro.5560020404.
A new method based on protein fragmentation and directly coupled microbore high-performance liquid chromatography-fast atom bombardment mass spectrometry (HPLC-FABMS) is described for determining the rates at which peptide amide hydrogens in proteins undergo isotopic exchange. Horse heart cytochrome c was incubated in D2O as a function of time and temperature to effect isotopic exchange, transferred into slow exchange conditions (pH 2-3, 0 degrees C), and fragmented with pepsin. The number of peptide amide deuterons present in the proteolytic peptides was deduced from their molecular weights, which were determined following analysis of the digest by HPLC-FABMS. The present results demonstrate that the exchange rates of amide hydrogens in cytochrome c range from very rapid (k > 140 h-1) to very slow (k < 0.002 h-1). The deuterium content of specific segments of the protein was determined as a function of incubation temperature and used to indicate participation of these segments in conformational changes associated with heating of cytochrome c. For the present HPLC-FABMS system, approximately 5 nmol of protein were used for each determination. Results of this investigation indicate that the combination of protein fragmentation and HPLC-FABMS is relatively free of constraints associated with other analytical methods used for this purpose and may be a general method for determining hydrogen exchange rates in specific segments of proteins.
本文描述了一种基于蛋白质片段化和直接联用微径高效液相色谱-快原子轰击质谱法（HPLC-FABMS）的新方法，用于测定蛋白质中肽酰胺氢进行同位素交换的速率。将马心脏细胞色素c在重水中随时间和温度进行孵育以实现同位素交换，转移至缓慢交换条件（pH 2 - 3，0℃）下，并用胃蛋白酶进行片段化处理。通过HPLC-FABMS对消化产物进行分析后测定蛋白水解肽段的分子量，从而推断出蛋白水解肽段中存在的肽酰胺氘原子数量。目前的结果表明，细胞色素c中酰胺氢的交换速率范围从非常快（k > 140 h⁻¹）到非常慢（k < 0.002 h⁻¹）。测定了蛋白质特定片段的氘含量随孵育温度的变化情况，并用于表明这些片段参与了与细胞色素c加热相关的构象变化。对于目前的HPLC-FABMS系统，每次测定大约使用5 nmol的蛋白质。本研究结果表明，蛋白质片段化与HPLC-FABMS相结合相对不受用于此目的的其他分析方法所带来的限制，可能是一种测定蛋白质特定片段氢交换速率的通用方法。
