Proteomic applications of protein quantification by isotope-dilution mass spectrometry

Over the decades, isotope-dilution mass spectrometry (IDMS) has been implemented extensively for accurate quantification of drugs, metabolites and peptides in body fluids and tissues. More recently, it has been extended for quantifying specific proteins in complex mixtures. In this extended methodology, proteins are subjected to endoprotease action and specific resultant peptides are quantified by using synthetic stable isotope-labeled standard (SIS) peptides and IDMS. This article outlines the utilities and applications of quantifying proteins by IDMS, emphasizing its complementary value to global survey-based proteomic studies. The potential of SIS peptides to provide quantitative insights into cell signaling is also highlighted, with specific examples. Finally, we propose several novel mass spectrometric data acquisition strategies for large-scale applications of IDMS and SIS peptides in systems biology and protein biomarker validation studies.     

An immunoaffinity liquid chromatography-tandem mass spectrometry assay for the quantitation of matrix metalloproteinase 9 in mouse serum

An immunoaffinity liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the quantitation of the zinc endopeptidase matrix metalloproteinase 9 (MMP-9) from mouse serum. Sample preparation for the assay included magnetic bead-based enrichment using an MMP-9 antibody and was performed in a 96-well plate format using a liquid-handling robotic platform. The surrogate peptide GSPLQGPFLTAR derived from MMP-9 by trypsin digestion was monitored using an on-line capillary flow trap-release chromatography setup incorporating a series of trap columns (C18, strong cation exchange, and another C18) prior to nanoflow chromatography and nanospray ionization with selected reaction monitoring (SRM) detection. The assay was fit-for-purpose validated and found to be accurate (<15% interbatch relative error) and precise (<15% interbatch coefficient of variation) across a range from 0.03 to 7.3 nM mouse MMP-9. Finally, the method was employed to measure MMP-9 concentrations in 30 naïve mouse serum samples, and results were compared with those obtained by an immunoassay.     

睾丸特异蛋白GGN1稳定表达细胞株的建立

GGN1是1种功能未知的睾丸特异表达蛋白,它是睾丸特异性基因Ggn的表达产物。Ggn基因的原初转录产物有多种剪接方式,并最终翻译产生3种蛋白,GGN1就是其中的1种。研究发现Ggn的表达与精子的生成过程紧密相关。GGN1还可以与多种蛋白相互作用,其中包括FANCL,GGNBP1,GGNBP2和OAZ3。为了能够在体外更方便的研究Ggn及其相关基因的功能,通过脂质体转染和G418加压法筛选获得了能稳定表达pEGFP-N2／GGN1的CHO—K1细胞株。     

A rapid screening method to monitor expression of recombinant proteins from various prokaryotic and eukaryotic expression systems using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry

Rapid methods using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry to monitor recombinant protein expression from various prokaryotic and eukaryotic cell culture systems were devised. Intracellular as well as secreted proteins from both induced and constitutive expression systems were measured and monitored from whole cells and growth media, thus providing an alternative to time-consuming traditional methods for screening and monitoring of protein expression. The methods described here involve minimal processing of samples and are therefore relevant to high-throughput screening applications.     

Expression in E. coli and purification of the nucleoside diphosphate kinase b from Leishmania major

Leishmaniasis is considered by the World Health Organization to be the second most important disease caused by a protozoan parasite. Biochemical and molecular biology studies can help in the understanding of the biology of the Leishmania parasite. All protozoan parasites, including Leishmania, are unable to synthesize purines de novo, and nucleoside diphosphate kinases (NDK) are involved in the salvage pathway by which free purines are converted to nucleosides and subsequently to nucleotides. In this report, we describe the cloning of NDK coding-sequence from Leishmania major, the expression of the enzyme containing a His(6)-tag in Escherichia coli, and purification of the catalytically active native protein by affinity chromatography using Ni-NTA resin.     

Proteomics for Protein Expression Profiling in Neuroscience

As the technology of proteomics moves from a theoretical approach to a practical reality, neuroscientists will have to determine the most appropriate applications for this technology. Neuroscientists will have to surmount difficulties particular to their research, such as limited sample amounts, heterogeneous cellular compositions in samples, and the fact that many proteins of interest are rare, hydrophobic proteins. This review examines protein isolation and protein fractionation and separation using two-dimensional electrophoresis (2-DE) and mass spectrometry proteomic methods. Methods for quantifying relative protein expression between samples (e.g., 2-DIGE, and ICAT) are also described. The coverage of the proteome, ability to detect membrane proteins, resource requirements, and quantitative reliability of different approaches is also discussed. Although there are many challenges in proteomic neuroscience, this field promises many rewards in the future.     

Application of high-precision isotope ratio monitoring mass spectrometry to identify the biosynthetic origins of proteins

Isotope ratio monitoring (IRM) mass spectrometry was used to measure the relative abundance of stable isotopes in several samples of adult human hemoglobin expressed in E. coli, yeast, and human blood. The results showed significant differences in the distribution of (15)N and (13)C isotopes among hemoglobin samples produced in these organisms. This indicates that IRM mass spectrometry can be used in forensic protein chemistry to identify the origin of protein expression.     

Role of tryptophan residues in the recognition of mutagenic oxidized nucleotides by human antimutator MTH1 protein

The human MTH1 antimutator protein hydrolyzes mutagenic oxidized nucleotides, and thus prevents their incorporation into DNA and any subsequent mutation. We have examined its great selectivity for oxidized nucleotides by analyzing the structure of the protein and its interaction with nucleotides, as reflected in the fluorescence of its tryptophan residues. The binding of nucleotides decreased the intensity of MTH1 protein fluorescence and red-shifted the emission peak, indicating that at least one tryptophan residue is close to the binding site. Oxidized nucleotides (2-OH-dATP and 8-oxo-dGTP) produced a larger decrease in fluorescence intensity than did unoxidized nucleotides, and MTH1 protein had a much higher binding affinity for oxidized nucleotides. Deconvolution of protein fluorescence by comparison of its quenching by positively (Cs(+)) and negatively (I(-)) charged ions indicated that the MTH1 tryptophan residues are in two different environments. One class of tryptophan residues is exposed to solvent but in a negatively charged environment; the other class is partially buried. While the binding of unoxidized nucleotides quenches the fluorescence of only class 1 tryptophan residue(s), the binding of oxidized nucleotides quenched that of class 2 tryptophan residue(s) as well. This suggests that selectivity is due to additional contact between the protein and the oxidized nucleotide. Mutation analysis indicated that the tryptophan residue at position 117, which is in a negative environment, is in contact with nucleotides. The negatively charged residues in the binding site probably correlate with the finding that nucleotide binding requires metal ions and depends upon their nature. Positively charged metal ions probably act by neutralizing the negatively charged nucleotide phosphate groups. (c) 2002 Elsevier Science Ltd.     

Mass spectrometric quantification of asparagine synthetase in circulating leukemia cells from acute lymphoblastic leukemia patients

The appearance of asparaginase-resistant acute lymphoblastic leukemia (ALL) in transformed cell lines has been correlated with increased expression of asparagine synthetase (ASNS). Recent measurements using mRNA-based assays have raised doubts, however, as to the importance of ASNS protein in the cellular mechanisms that confer drug resistance upon the leukemic cells. Studies aimed at determining the concentration of ASNS protein in human leukemias are therefore needed to resolve this issue. A mass spectrometry (MS)-based procedure is presented for the direct quantification of ASNS protein concentration in complex sample mixtures. This assay is able to distinguish samples from transformed cell lines that express ASNS over a wide dynamic range of concentration. Importantly, this method directly detects ASNS protein, the functional entity that may be synthesizing sufficient asparagine to render leukemia cells resistant to asparaginase-treatment. We also report the successful use of this MS method, which has lower limits of detection and quantification of 30 and 100 attomoles, respectively, for the first direct measurements of ASNS protein concentrations in four patient blast samples.     

化学交联质谱技术的研究进展

化学交联质谱技术是解析蛋白质结构和研究蛋白质相互作用的重要工具。近5年以来,该技术在方法和应用上都取得了很大的进步。方法上,一方面可断裂交联剂与新型分离富集方法展现了较好的应用前景,另一方面更加高效的交联肽段搜索引擎和质量控制方法为交联质谱数据分析提供了有力的工具。应用上,一方面与冷冻电镜技术结合解析了大量蛋白质的结构,另一方面从研究蛋白质复合物的相互作用发展到研究全蛋白质组水平的相互作用网络。化学交联质谱技术在方法和应用上的蓬勃发展,体现了这一技术的重要作用。本文对化学交联质谱技术的各个环节进行了详细的综述,包括交联剂选择、交联反应、酶切、交联肽段富集、液质联用、交联肽段鉴定、质量控制和生物学应用,重点介绍了最近5年的研究进展。最后,讨论了化学交联质谱技术面临的挑战及未来的发展方向。     

DNA 修复酶 MTH1 抑制剂研究进展

MTH1 是一种 DNA 氧化损伤修复酶,主要负责“清理”核苷酸池中氧化损伤的脱氧核苷三磷酸(dNTPs),以防其掺入 DNA 复 制中而造成碱基错配。研究表明,MTH1 与肿瘤细胞的生存密切相关,而正常细胞的生长与存活则不依赖于 MTH1。所以,以 MTH1 为靶 点开展抗肿瘤新药研发,已逐渐受到人们的关注。抑制 MTH1,为肿瘤治疗开辟了一条新途经。简介 MTH1 的结构和功能及其与肿瘤的关联, 着重对近年来 MTH1 抑制剂的发现过程和研究进展作一综述,探究小分子 MTH1 抑制剂与 MTH1 蛋白的作用模式,为 MTH1 抑制剂的设 计提供思路。     

蛋白质结构与相互作用研究新方法——交联质谱技术

蛋白质的空间结构信息以及蛋白质间的相互作用信息对于研究蛋白质的功能有重要意义.研究蛋白质结构与相互作用的传统技术,如核磁共振技术、X射线晶体衍射技术等,对于蛋白质的纯度、结晶性和绝对量均有比较高的要求,限制了其广泛应用.交联质谱技术是近十多年来发展起来的新技术,它将质谱技术与交联技术相结合,在研究蛋白质结构与相互作用方面具有速度快、成本小、蛋白质各方面性状要求低等优势.本文就交联质谱技术各个环节的技术方法加以综述,包括交联质谱实验分离富集技术、常见交联剂特性、交联质谱数据库搜索算法、结果验证研究和交联质谱技术的应用等方面,并展望了该研究方向未来的发展.     

Proteomic Profiling and Neurodegeneration in Alzheimer's Disease

Quantitative proteome analysis of Alzheimer's disease (AD) brains was performed using 2-D gels to identify disease specific changes in protein expression. The task of characterizing the proteome and its components is now practically achievable because of the development and integration of four important tools: protein, EST, and complete genome sequence databases, mass spectrometry, matching software for protein sequences and protein separation technology. Mass spectrometry (MS) instrumentation has undergone a tremendous change over the past decade, culminating in the development of highly sensitive, robust instruments that can reliably analyze biomolecules, particularly proteins and peptides; we identified 35 proteins from over 100 protein spots on a 2-D gel. Using this current technology, protein-expression profiling, which is actually a specialized form of mining, is an important principal application of proteomics. The information obtained has tremendous potential as a means of determining the pathogenesis, and detecting disease markers and potential targets for drug therapy in AD.     

“De novo” sequencing of peptides recovered from in-gel digested proteins by nanoelectrospray tandem mass spectrometry

Proteins separated by one-dimensional or two-dimensional gel electrophoresis can be digested in-gel with trypsin and the recovered peptides can be sequenced de novo using triple quadrupole or hybrid quadrupole time-of-flight instruments equipped with a nanoelectrospray ion source. The peptide sequences determined provide useful information for identification of proteins by homology searching for cloning of the cognate genes by PCR based approaches.     

Transmembrane and secreted MUC1 probes show trafficking-dependent changes in O-glycan core profiles

The human mucin MUC1 is expressed both as a transmembrane heterodimeric protein complex that recycles via the trans-Golgi network (TGN) and as a secreted isoform. To determine whether differences in cellular trafficking might influence the O-glycosylation profiles on these isoforms, we developed a model system consisting of membrane-bound and secretory-recombinant glycosylation probes. Secretory MUC1-S contains only a truncated repeat domain, whereas in MUC1-M constructs this domain is attached to the native transmembrane and cytoplasmic domains of MUC1 either directly (M0) or via an intermitting nonfunctional (M1) or functional sperm protein-enterokinase-agrin (SEA) module (M2); the SEA module contains a putative proteolytic cleavage site and is associated with proteins receiving extensive O-glycosylation. We showed that MUC1-M2 simulates endogenous MUC1 by recycling from the cell surface of Chinese hamster ovary (CHO) mutant ldlD14 cells through intracellular compartments where its glycosylation continues. The profiles of O-linked glycans on MUC1-S secreted by epithelial EBNA-293 and MCF-7 breast cancer cells revealed patterns dominated by core 2-based oligosaccharides. In contrast, the respective membrane-shed probes expressed in the same cells showed a complete shift to patterns dominated by sialyl core 1. In conclusion, glycan core profiles reflected the subcellular trafficking pathways of the secretory or membranous probes and the modifying activities of the resident glycosyltransferases.     

Comparison of folate quantification in foods by high-performance liquid chromatography-fluorescence detection to that by stable isotope dilution assays using high-performance liquid chromatography-tandem mass spectrometry

A comparison study on folate quantitation was carried out between the recently developed stable isotope dilution assay using liquid chromatography-tandem mass spectrometry (LC-MS-MS) and the frequently used HPLC with fluorimetric detection (LC-FD). By applying LC-MS-MS, spinach, wheat bread, beef, and blood plasma were found to contain 159.2, 19.8, 1.2, and 5.6 microg/100 g total folates, respectively, whereas the respective quantitative data obtained by LC-FD were 95.5, 16.2, 0.7, and 6.8 microg/100 g. In all samples, LC-MS-MS revealed superior selectivity and precision and circumvented the shortcomings of conventional LC techniques, i.e., ambiguous peak assignment as well as high detection limits for 5-formyltetrahydrofolate, 10-formylfolic acid, and folic acid. The affinity chromatography columns used in this study showed excellent cleanup performance and permitted detection limits as low as 0.1, 0.5, 0.1, 0.08, and 0.1 microg/100 g for tetrahydrofolate (H(4)folate), 5-methyl-H(4)folate, 5-formyl-H(4)folate, 10-formylfolate, and pteroylglutamic acid, respectively. Thus, a 10-fold higher sensitivity compared to solid-phase anion-exchange cartridges was achieved. However, affinity chromatography columns revealed a significantly higher affinity toward the natural vitamers than to the racemic isotopomeric standards, which has to be considered when applying the latter in stable isotope dilution assays.