PAS-cal: a Generic Recombinant Peptide Calibration Standard for Mass Spectrometry
PAS-cal: a Generic Recombinant Peptide Calibration Standard for Mass Spectrometry
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DOI：
10.1007/s13361-014-0902-3
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发表时间：
2014-08-01
影响因子：
3.2
通讯作者：
Skerra, Arne
中科院分区：
文献类型：
--
作者：
Breibeck, Joscha;Serafin, Adam;Skerra, Arne
我们描述了一种新的具有均匀生物物理和电离特性的重组多肽标准物质的设计、制备和质谱学表征。PAS-CAL是一种人工合成的多肽链接物，由不同长度的多肽盒组成，每个多肽盒由三个化学稳定的小氨基酸Pro、Ala和Ser组成，中间夹杂着Arg残基，允许与胰酶进行位点特异性切割。Pas-cal在大肠杆菌中以一种小的泛素样修饰物(SUMO)融合蛋白的形式高效表达，该融合蛋白易于纯化，并允许在SUMO蛋白酶切割后分离Pas-A部分。随后用胰酶原位处理后，该多肽产生一组4个定义的均一肽，大小在2到8 kDa之间，质量间距相等。ESI-MS分析显示了一个易于解释的原始光谱，经过去卷积后，得到了非常简单的四个峰的模式，具有相似的电离信号。MALDI-MS分析包括完整多肽及其胰酶片段的多肽混合物，不仅揭示了四个标准多肽，而且揭示了完整级联分子的单电荷和双电荷状态以及多肽之间的二聚体和三聚体加合离子物种，从而扩大了可观察到的m/z范围。PAS-CAL的有利性质很可能是由于PAS序列的强烈亲水性和构象无序的类似聚乙二醇的性质的结果。因此，PAS-CAL为蛋白质/多肽生物分析和蛋白质组学研究中的质谱学提供了一个廉价和通用的重组多肽校准标准，其组成可以进一步调整以适应个人需求。
We describe the design, preparation, and mass-spectrometric characterization of a new recombinant peptide calibration standard with uniform biophysical and ionization characteristics for mass spectrometry. "PAS-cal" is an artificial polypeptide concatamer of peptide cassettes with varying lengths, each composed of the three small, chemically stable amino acids Pro, Ala, and Ser, which are interspersed by Arg residues to allow site-specific cleavage with trypsin. PAS-cal is expressed at high yields in Escherichia coli as a Small Ubiquitin-like MOdifier (SUMO) fusion protein, which is easily purified and allows isolation of the PAS-cal moiety after SUMO protease cleavage. Upon subsequent in situ treatment with trypsin, the PAS-cal polypeptide yields a set of four defined homogeneous peptides in the range from 2 to 8 kDa with equal mass spacing. ESI-MS analysis revealed a conveniently interpretable raw spectrum, which after deconvolution resulted in a very simple pattern of four peaks with similar ionization signals. MALDI-MS analysis of a PAS-cal peptide mixture comprising both the intact polypeptide and its tryptic fragments revealed not only the four standard peptides but also the singly and doubly charged states of the intact concatamer as well as di- and trimeric adduct ion species between the peptides, thus augmenting the observable m/z range. The advantageous properties of PAS-cal are most likely a result of the strongly hydrophilic and conformationally disordered PEG-like properties of the PAS sequences. Therefore, PAS-cal offers an inexpensive and versatile recombinant peptide calibration standard for mass spectrometry in protein/peptide bioanalytics and proteomics research, the composition of which may be further adapted to fit individual needs.