Methods In Mass Spectrometry

用于分析蛋白质结构的质谱方法正在蛋白质组学的独立和合作研究项目中得到改进、应用和测试。分离方法和数据处理方面的项目正在进行中。在过去一年中，在以下方面取得了进展：实施质谱蛋白质组学数据处理的计算环境;同位素标记的融合蛋白作为蛋白质标准品;对复杂的液相色谱-高分辨率质谱数据中已鉴定和未鉴定的组分进行定量;以及对蛋白质组学标准进行多机构合作研究。 质谱蛋白质组学数据的处理和挖掘需要一个系统，允许研究人员台式计算机访问TB级的数据;能够使用新兴的软件工具反复探测他们的数据;并能够将这些数据转移到公共档案库。 一个多功能的系统已经在LNT中实现，并将与NIH的其他蛋白质组学小组共享。 我们试图确定生物合成的串联标记肽（多联体）是否等同于整个标记的蛋白质作为同位素稀释质谱法的内标物，使用三重四极杆质量分析仪上的选定反应监测。 质谱法通过使用多反应监测来跟踪肽片段的特定转变，为这些测量提供了平台;然而，内标物只有在忠实地模拟全长蛋白质的蛋白水解特性时才是准确的。 我们通过质谱筛选以及公共数据库和文献选择了签名肽加上12个氨基酸（6个氨基末端和6个羧基末端）。 将延伸的选定序列的合成基因与亲和标签融合，并通过将合成基因克隆到表达载体中并使用13 C和15 N精氨酸和赖氨酸氨基酸标记来表达。 人血清白蛋白（HSA）多联体进行了测试，因为天然HSA是容易获得的，以及15 N标记的全长HSA作为实验室标准。 使用标准曲线测量相对于化学合成的链球菌标签10-mer肽的HSA多联体浓度。 时间，温度和酶的研究进行了优化。 HSA多联体中的五种肽中的三种准确地模拟天然HSA的胰蛋白酶性质。 我们证明了串联的HSA肽可以用作尿样品中HSA定量的内标。 传统的免疫比浊法数据提供了可比的结果。 对于免疫比浊测定，消耗50微升尿液;对于所选的反应监测质谱法，消耗少于5微升。

Mass spectrometric methods for the analysis of protein structures are being improved, applied and tested in independent and collaborative research projects in proteomics. Projects in separation methods and data processing are in progress. In the past year, progress has been made on implementation of a computational environment for mass spectrometric proteomic data processing; isotope labeled fusion proteins as protein standards; quantification of identified and unidentified components in sets of complex liquid chromatography-high resolution mass spectrometry data; and multi-institutional collaborative studies on standards in proteomics. The processing and mining of mass spectrometric proteomic data requires a system allowing investigators desktop computer access to terabytes of data; the ability to repeatedly probe their data with emerging software tools; and the ability to transfer this data to public archival repositories. A versatile system has been implemented in LNT and will be shared with other proteomics groups in NIH. We sought to determine if biosynthetic concatenated labeled peptides (concatemers) are equivalent to whole labeled proteins as internal standards for isotope dilution mass spectrometry using selected reaction monitoring on a triple quadrupole mass analyzer. Mass spectrometry provides a platform for these measurements by using multiple reaction monitoring to follow specific transitions of peptides as they fragment; however, internal standards are only accurate if they faithfully mimic proteolytic properties of full-length proteins. We selected signature peptides plus 12 amino acids (6 amino- and 6 carboxy-terminal) through mass spectrometric screening as well as the public databases and literature. Synthetic genes for the extended selected sequences are fused with affinity tags and expressed by cloning a synthetic gene into an expression vector and labeled using 13C and 15N arginine and lysine amino acids. A human serum albumin (HSA) concatemer was tested because native HSA is readily available as well as 15N-labeled full-length HSA as a laboratory standard. HSA concatemer concentration was measured with respect to a chemically synthesized strep tag 10-mer peptide using a standard curve. Time, temperature, and enzyme studies were optimized. Three of the five peptides in the HSA concatemer accurately mimic the tryptic properties of native HSA. We demonstrated that concatenated HSA peptides can be used as internal standards for the quantification of HSA in urine samples. Traditional immunoturbidimetric data provided comparable results. For the immunoturbidimetric assays, 50 microliters of urine are consumed; for the selected reaction monitoring mass spectrometry method, less than 5 microliters are consumed.