Qian Yuwei, Preston Ken, Krokhin Oleg, Mellish Jean, Ens Werner
Department of Physics and Astronomy, University of Manitoba, Winnipeg, Manitoba, Canada.
J Am Soc Mass Spectrom. 2008 Oct;19(10):1542-50. doi: 10.1016/j.jasms.2008.06.008. Epub 2008 Jun 27.
We have performed a detailed characterization and identification of wheat gluten proteins obtained from the Teal variety of Canadian hard red spring wheat. RP-HPLC separation of the sample into 35 fractions has reduced the spectral complexity; this was followed by MALDI mass spectrometry (MS), which showed the presence of six or fewer resolved protein components above 20 kDa in each RP-HPLC fraction, giving a total of 93 MS resolved peaks. These included 17 peaks in the omega-gliadin fractions (F1-4), 12 in the high molecular weight (HMW) glutenin subunit fractions (F5-8), 59 in the alpha- and beta-gliadins and low molecular weight (LMW) glutenin subunit fractions (F9-31) and 5 peaks in the gamma-gliadin fractions (F32-35). Peptide maps of tryptic digests of HPLC fractions were obtained from a tandem quadrupole time-of-flight mass spectrometer (MALDI QqTOF MS) and were submitted to the ProFound search engine. HMW glutenin subunits including Ax2*, Dx5, Bx7, and Dy10 (consistent with the known profile of Teal), and LMW glutenin subunits including six from group 3 type II and 1 from group 2 type I, were identified with reasonable sequence coverage from HPLC fraction 5, 7, 17, and 18. The identities of the peptides attributed to selected gluten proteins were confirmed using MS/MS with BioMultiView to match the predicted and measured partial amino acid sequences. Because of incomplete wheat DNA databases, many wheat gluten proteins could not be identified. These results suggest that the combination of RP-HPLC with MS and MS/MS techniques is a promising approach for the characterization of wheat gluten proteins.
我们对从加拿大硬红春小麦蒂尔品种中获得的小麦面筋蛋白进行了详细的表征和鉴定。通过反相高效液相色谱（RP-HPLC）将样品分离为35个组分，降低了光谱复杂性；随后进行基质辅助激光解吸电离质谱（MALDI-MS）分析，结果表明每个RP-HPLC组分中分子量高于20 kDa的可分辨蛋白质组分有6个或更少，总共得到93个质谱可分辨峰。其中包括ω-醇溶蛋白组分（F1-4）中的17个峰、高分子量（HMW）谷蛋白亚基组分（F5-8）中的12个峰、α-和β-醇溶蛋白以及低分子量（LMW）谷蛋白亚基组分（F9-31）中的59个峰和γ-醇溶蛋白组分（F32-35）中的5个峰。通过串联四极杆飞行时间质谱仪（MALDI QqTOF MS）获得了HPLC组分胰蛋白酶消化产物的肽图，并将其提交给ProFound搜索引擎。从HPLC组分5、7、17和18中鉴定出了包括Ax2*、Dx5、Bx7和Dy10（与蒂尔的已知谱型一致）的HMW谷蛋白亚基，以及包括6个来自第3组II型和1个来自第2组I型的LMW谷蛋白亚基，序列覆盖率合理。使用BioMultiView通过串联质谱（MS/MS）确认了归因于选定面筋蛋白的肽的身份，以匹配预测和测量的部分氨基酸序列。由于小麦DNA数据库不完整，许多小麦面筋蛋白无法鉴定。这些结果表明，RP-HPLC与MS和MS/MS技术相结合是表征小麦面筋蛋白的一种有前景的方法。
