Characterization Of Proteins By Mass Spectrometry

我们与NICHD的团队合作，对蛋白质的质谱表征进行研究，作为我们的首要任务，但我们也对质谱蛋白质表征进行独立研究。这项工作的一个主要方面是鉴定在其他PI的生化研究中分离的蛋白质。 在未知蛋白质的鉴定方面，MS数据用于查询基因组数据库以提出一般性问题，“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）质谱分析，液相色谱（LC），然后在能够使用裂解反应生成肽序列的仪器（即LC-MS/MS）中进行电喷雾电离和质谱分析，以及MALDI，然后进行串联TOF分析，用于根据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，我们已经开始解决的问题，提供序列信息的蛋白质没有在数据库中描述，由于数据库错误或不完整性，这种不完整性是最常见的生物体具有未知或部分特征的基因组。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，并需要使用MALDI串联TOF。当MALDI串联TOF光谱与软件进行分析，我们已经编写了这项任务，我们的测序能力代表了一个独特的能力，在美国国立卫生研究院，并为大多数实验室做质谱表征的蛋白质。我们的程序依赖于亚稳态肽离子的分解，没有随后的碰撞，在第一个质量分析仪之后，但在所谓的“碰撞池”之前，通过选择候选离子建立的时间范围内。该方法产生了非常可靠的序列多达7个肽的范围内的7至18个残基从胰蛋白酶蛋白水解的蛋白质分析为未知的;在30 kD的蛋白质的情况下，这将是一个绝对确定的约30%的蛋白质的整个序列。在过去，这种方法的局限性是由于近同量异位素干扰，即，不能被质量选择电子器件分辨的前体离子质量，其导致由多个前体离子产生的碎裂谱，并且因此是不可解释的。使用最近安装的样品点样机器人，使空间分离的LC梯度到MALDI靶板应减少这些干扰的数量，从而允许更广泛的覆盖绝对序列。 在与完全从头测序工作密切相关的领域，我们开发并实施了软件，提高了我们确定某些类型肽的能力：那些含有翻译后修饰的肽和那些由不常用的蛋白酶（特别是胃蛋白酶）产生的肽。此外，我们已经取得了实质性的进展，在磷酸化位点的检测使用差分MALDI光谱的方法，比较正离子和负离子光谱。该方法使用甲醇HCl酯化羧酸位点;酯化的酸性残基在负离子MALDI中不能有效地降解，但磷酸化肽不受影响。 在过去的一年里，我们还开始了一个项目，用于表征早产患者羊水的蛋白质质量指纹。这项研究的假设是，质谱可以用来区分早产导致早产，从这样的劳动，不导致早产。正在开发的方法采用从稀释的羊水样品中获得的2- 20 kDa范围内的MALDI质谱的比较，所述羊水样品已经脱盐，然后直接应用于MALDI样品阶段。我们已经开发了一个实验设计，使我们能够表征这些光谱的变化所产生的各种参数在实验中。我们在MatLab中开发了一种数学/统计方法，可以自动进行ANOVA和主成分分析，并可靠地区分样本类别。初步结果表明，我们能够区分不同患者组的羊水来源。

We conduct research on the mass spectrometric characterization of proteins both collaboratively with groups in NICHD as our first priority, but we also conduct independent investigations in mass spectrometric protein characterization. A major aspect of this work is the identification of proteins isolated in biochemical investigations of other PIs. In terms of 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 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; this incompleteness is associated most frequently 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 and requires the use of a MALDI tandem TOF. When the MALDI tandem TOF spectra are analyzed with software we have written for this task, our capability for sequencing represents a unique capability at NIH, and for most laboratories doing mass spectrometric characterization of proteins. Our procedure relies on the decomposition of metastable peptide ions, without subsequent collisions, in the time frame of the established by the selection of candidate ions after the first mass analyzer, but prior to the so-called "collision cell". The method yields very reliable sequences for as many as seven peptides in the range of 7 to 18 residues taken from the tryptic proteolysis of proteins analyzed as unknowns; in the case of a 30kD protein, this would amount to an absolute determination of about 30% of the entire sequence of the protein. In the past, the limitation of this approach has been due to near-isobaric interferences,i.e., precursor ion masses that cannot be resolved by the mass selection electronics, that lead to fragmentation spectra arising from multiple precursor ions, and re thus unintepretable. The use of a recently installed sample-spotting robot to enable spatial separations of LC gradients onto a MALDI target plate should reduce the number of these interferences and thus allow more extensive coverage of absolute sequences. In a area very closely related to the Complete de Novo Sequencing work, we have developed and implemented software that increases our ability to pinpoint certain types of peptides: those containing post-translational modifications and those arising from less commonly used proteases, particularly pepsin. In addition, we have made substantial progress in the detection of phosphorylation sites using a differential MALDI spectra approach that compares positive and negative ions spectra. The method employs the esterification of carboxylic acid sites using methanolic HCl; the esterified acidic residues do not ionize efficiently in negative ion MALDI, but the phosphorylated peptides are unaffected. Within the past year we have also begun a project for characterizing the protein mass fingerprints of amniotic fluid from patients who have undergone premature labor. The hypothesis of the investigation is that mass spectra can be used to differentiate premature labor leading to pre-term delivery from such labor that does not result in pre-term delivery. The methodology under development employs comparisons of MALDI mass spectra in the range of 2-20kDa obtained from samples of diluted amniotic fluid samples that have been desalted and then applied directly to the MALDI sample stage. We have developed an experimental design that allows us to characterize the variance of these spectra arising from a variety of parameters in the experiment. We have developed a mathematical/statistical approach in MatLab to automate both ANOVA and Principal Component Analysis and reliably differentiate between classes of samples. Preliminary results show that we are able to differentiate between the sources of amniotic fluid in groups of patients.