Characterization Of Proteins By Mass Spectrometry

我们与NICHD的团队合作进行蛋白质的质谱学表征研究是我们的首要任务，但我们也进行独立的蛋白质质谱学研究。这项工作的一个主要方面是鉴定在其他PI的生化研究中分离出来的蛋白质。 在未知蛋白质的鉴定方面，MS数据被用来查询基因组数据库，以提出一个一般性的问题：数据库中存在的蛋白质序列中是否有预期的蛋白水解性裂解产物具有与从未知蛋白质中产生的经验确定的多肽质量相匹配的理论质量？有三种质谱学方法可用于这项工作。基质辅助激光解吸电离(MALDI)与飞行时间(TOF)质量分析，在能够利用裂解反应产生肽序列的仪器中，即LC-MS/MS中，高效液相(LC)和电喷雾电离(LC)，以及MALDI和串联飞行时间(TOF)分析，用于从碎片离子光谱中确定肽序列。有了这种仪器的组合，我们相信，给出足够的凝胶带中的物质，允许多达100 fmole可用于分析，可以对数据库中描述的蛋白质做出积极的鉴定。 为了提高蛋白质表征能力，正在进行几个领域的开发。首先，我们已经开始解决由于数据库错误或不完整而提供数据库中未描述的蛋白质的序列信息的问题；这种不完整最常与具有未知或部分特征的基因组的生物体有关。我们正在采取一种我们称之为“完整的多肽从头测序”的方法。与其他广泛使用的方法相比，这种方法是新颖的，在这些方法中，找到了所谓的肽的“序列标签”。序列标签包括从肽裂解质谱中确定2到5个氨基酸残基以及亲本质量以搜索数据库。我们的方法需要确定整个多肽的氨基酸序列，并需要使用MALDI串联TOF。当用我们为这项任务编写的软件分析MALDI串联飞行时间谱时，我们的测序能力代表了NIH的独特能力，对于大多数对蛋白质进行质谱分析的实验室来说。我们的程序依赖于亚稳态多肽离子的分解，在没有后续碰撞的时间框架内，在第一次质量分析器之后，但在所谓的“碰撞池”之前，通过选择候选离子来建立。该方法产生了多达7个多肽的非常可靠的序列，这些多肽取自分析为未知的蛋白质的胰酶蛋白分解的7到18个残基；对于30kD的蛋白质，这将相当于绝对确定大约30%的整个蛋白质序列。过去，这种方法的局限性是由于近等压干扰，即质量选择电子学不能分辨的前体离子质量，导致多个前体离子产生碎裂光谱，因此无法解释。使用最近安装的样品点样机器人能够在MALDI靶板上实现LC梯度的空间分离，应该会减少这些干扰的数量，从而允许更广泛地覆盖绝对序列。 在与完整的De Novo测序工作密切相关的一个领域，我们已经开发并实施了软件，以提高我们定位特定类型多肽的能力：那些包含翻译后修饰的多肽，以及那些由不太常用的蛋白酶产生的多肽，特别是胃酶。此外，我们在使用比较正离子和负离子光谱的差示MALDI光谱方法检测磷酸化位点方面取得了实质性进展。该方法使用甲醇盐酸对羧酸中心进行酯化，酯化的酸性残基在负离子MALDI中不能有效电离，但磷酸化的多肽不受影响。 在过去的一年里，我们还开始了一个项目，以确定早产患者羊水的蛋白质质量指纹。这项研究的假设是，质谱学可以用来区分导致早产的早产和不会导致早产的早产。正在开发的方法使用从稀释的羊水样品中获得的2-20 kDa范围内的MALDI质谱图的比较，这些样品经过脱盐，然后直接应用于MALDI样品台。我们已经开发了一种实验设计，允许我们表征这些光谱在实验中因各种参数而产生的变化。我们在MatLab中开发了一种数学/统计方法来自动化ANOVA和主成分分析，并可靠地区分样本类别。初步结果显示，我们能够区分不同组患者的羊水来源。

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.