Characterization Of Proteins By Mass Spectrometry

这项工作的主要目标是使用质谱法在微尺度水平上识别和详细表征蛋白质和肽。为了鉴定未知蛋白质，MS数据用于查询基因组数据库以询问一般问题，“数据库中存在的任何蛋白质序列是否具有预期的蛋白水解切割产物，其理论质量与由未知产生的肽的经验确定的质量相匹配？“三种质谱方法可用于这项工作。基质辅助激光解吸电离（MALDI）和飞行时间（TOF）质量分析，液相色谱（LC），然后在能够使用裂解反应生成肽序列的仪器中进行电喷雾电离和质量分析，即LC-MS/MS，以及最近的MALDI，然后串联TOF分析，用于从碎片离子光谱测定肽序列。有了这种仪器组合，我们相信，如果凝胶带中有足够的材料，允许多达100 fmole可用于分析，则可以对数据库中描述的蛋白质进行阳性鉴定。 为了提高蛋白质表征能力，有几个发展领域正在进行。首先，我们已经开始解决提供数据库中没有描述的蛋白质序列信息的问题，由于数据库错误或不完整，最常见的是与具有未知或部分特征的基因组的生物体相关。我们正在采取我们称之为“肽的完全从头测序”的方法。与其他广泛使用的方法相比，这种方法是新颖的，其中发现了所谓的肽的“序列标签”。序列标签包括从肽片段质谱中确定2至5个氨基酸残基，沿着母体质量以搜索数据库。我们的方法需要确定整个肽的氨基酸序列。这种方法仅限于使用MALDI串联TOF，并且在这方面是NIH独有的能力。最近的结果，利用最近升级的MALDI串联TOF仪器，表明我们的程序产生非常可靠的序列多达7个肽的范围内的8至17个残基从胰蛋白酶水解的海胆蛋白。我们能够在串联TOF中采用后续的碰撞诱导解离（CID）实验。在这些情况下，在第一个实验中确定的序列不仅可以被验证，而且还允许Leu残基与Ile区分开。使用本节中设计和编写的新型软件，对该方法所需的肽的单个裂解质谱进行详细解释。 在两个密切相关的领域，我们正在开发两种类型的翻译后修饰，磷酸化和二硫键的鉴定方法。这两种方法的开发涉及使用多个质谱实验和使用本节中编写的新软件。这两种类型的问题是复杂的，在一个不同的方式比那些涉及序列测定，因为在这些情况下被研究的蛋白质必须有已知的序列。为了有效地确定修饰，必须使用消化和质谱法获得蛋白质的几乎完全的肽覆盖。磷酸化位点鉴定首先涉及使用金属亲和色谱富集/分离磷酸肽，然后结合质谱。通过正负离子MALDI TOF谱之间的离子强度差异发现潜在的磷酸肽。然后使用LC-MS/MS或串联TOF方法从经历中性磷酸盐损失的氨基酸残基中鉴定磷酸化位点。鉴定二硫桥的位点也是以类似的方式进行的。比较从还原和未还原蛋白质获得的蛋白水解肽的MADLI TOF谱，并对还原物质谱中不存在于未还原物质中的肽进行测序。然后研究具有未还原物质特有质量的这些肽的潜在配对。

The principal goal in this work is the identification and detailed characterization of proteins and peptides at a microscale level using mass spectrometry. For identification of unknown proteins, the MS data are used to query genomic databases to ask the general question, "Do any of the protein sequences present in the data base have expected proteolytic cleavage products with theoretical masses that match the empirically determined masses of the peptides generated from the unknown?" Three mass spectrometric approaches are available for this effort. Matrix Assisted Laser Desorption Ionization (MALDI) with Time-of-Flight (TOF) mass analysis, liquid chromatography (LC) followed by electrospray ionization with mass analysis in an instrument capable of using fragmentation reactions to generate peptide sequences, i.e. LC-MS/MS, and most recently MALDI followed by tandem TOF analysis for the determination of peptide sequences from fragment ion spectra. With this combination of instrumentation, we are confident that, given enough material in a gel band to allow as much as 100 fmole to be available for analysis, a positive identification can be made for a protein that is described in a database. There are several areas of development that are being followed in order to improve protein characterization capabilities. First, we have begun addressing the question of providing sequence information on proteins that are not described in data bases, due to data base error or incompleteness, most frequently associated with organisms having unknown or partially characterized genomes. We are taking the approach we have termed "Complete de novo Sequencing of Peptides". This approach is novel in comparison to the other widely used methods, in which the so-called "sequence tag" for a peptide is found. The sequence tag consists of determining between 2 and 5 amino acid residues from a peptide fragmentation mass spectrum along with the parent mass to search a database. Our approach requires the determination of the amino acid sequence of the entire peptide. This approach is limited to the use of a MALDI tandem TOF, and in that regard is a capability unique at NIH. Recent results, making use of the recently upgraded MALDI tandem TOF instrument, show that our procedure yields very reliable sequences for as many as seven peptides in the range of 8 to 17 residues taken from the tryptic proteolysis of sea urchin proteins. We are able to employ subsequent collision induced dissociation (CID) experiment in the tandem TOF. Under those circumstances, a sequence determined in the first experiment can not only be verified, but also allow Leu residues to be distinguished from Ile. The detailed interpretation of individual fragmentation mass spectra of peptides required for this approach is being implemented using novel software designed and written in this Section. In two closely related areas, we are developing methods both for the identification of two types of post-translational modifications, phosphorylation and disulfide links. The development of both of these methodologies involves the use of multiple mass spectrometry experiments and the use of novel software written in this Section. These two types of problems are complex in a different fashion than those involving sequence determination since the proteins being investigated in these cases must have known sequences. In order to determine the modifications effectively, virtually complete peptide coverage of the proteins must be obtained using digestion and mass spectrometry. Phosphorylation site identification involves first the enrichment/isolation of phosphopeptides using metal affinity chromatography followed by a combination of mass spectrometry. Potential phosphopeptides are found by differences in ion intensities between positive and negative ion MALDI TOF spectra. Phosphorylation sites are then identified from amino acids residues undergoing neutral phosphate losses in using LC-MS/MS or tandem TOF approaches. Identifying the sites of disulfide bridges is done in a somewhat similar fashion. MADLI TOF spectra of proteolytic peptides obtained from reduced and unreduced proteins are compared and peptides in the spectrum of the reduced material that are not present in the unreduced are sequenced. Potential pairing of those peptides with masses that are unique to the unreduced material is then investigated.