Showing papers by "Jimmy K. Eng published in 2003"

The study of macromolecular complexes by quantitative proteomics.

Abstract: We describe a generic strategy for determining the specific composition, changes in the composition, and changes in the abundance of protein complexes. It is based on the use of isotope-coded affinity tag (ICAT) reagents 1 and mass spectrometry to compare the relative abundances of tryptic peptides derived from suitable pairs of purified or partially purified protein complexes. In a first application, the genuine protein components of a large RNA polymerase II (Pol II) preinitiation complex (PIC) were distinguished from a background of co-purifying proteins by comparing the relative abundances of peptides derived from a control sample and the specific complex that was purified from nuclear extracts by a single-step promoter DNA affinity procedure 2 . In a second application, peptides derived from immunopurified STE12 protein complexes isolated from yeast cells in different states were used to detect quantitative changes in the abundance of the complexes, and to detect dynamic changes in the composition of the samples. The use of quantitative mass spectrometry to guide identification of specific complex components in partially purified samples, and to detect quantitative changes in the abundance and composition of protein complexes, provides the researcher with powerful new tools for the comprehensive analysis of macromolecular complexes.

Quantitative proteomic analysis indicates increased synthesis of a quinolone by Pseudomonas aeruginosa isolates from cystic fibrosis airways

Abstract: The opportunistic bacterial pathogen Pseudomonas aeruginosa colonizes airways of individuals with cystic fibrosis (CF) with resultant chronic destructive lung disease. P. aeruginosa adaptation to the CF airway includes biofilm formation and antibiotic resistance. Isolates from asymptomatic individuals in the first 3 years of life have unique characteristics, suggesting that adaptation occurs before clinical symptoms. One defined early adaptation is expression of a specific proinflammatory lipopolysaccharide (LPS) that is associated with antimicrobial peptide resistance. This CF-specific LPS is induced when P. aeruginosa is grown in medium that is limited for magnesium. Therefore, qualitative and quantitative proteomic approaches were used to define 1,331 P. aeruginosa proteins, of which 145 were differentially expressed on limitation of magnesium. Among proteins induced by low magnesium were enzymes essential for production of 2-heptyl 3-hydroxy 4-quinolone, the Pseudomonas quinolone signal (PQS), which interacts with the homoserine lactone signaling pathway. Measurement of PQS in P. aeruginosa isolates from asymptomatic children with CF indicated that strains with increased synthesis of PQS are present during early colonization of CF patient airways.

Identification of androgen-coregulated protein networks from the microsomes of human prostate cancer cells

Abstract: Background: Androgens play a critical role in the development of prostate cancer-dysregulation of androgen-regulated growth pathways can led to hormone-refractory prostate cancer. A comprehensive understanding of androgen-regulated cellular processes has not been achieved to date. To this end, we have applied a large-scale proteomic approach to define cellular processes that are responsive to androgen treatment in LNCaP prostate cancer cells. Results: Using isotope-coded affinity tags and mass spectrometry we identified and quantified the relative abundance levels of 1,064 proteins and found that distinct cellular processes were coregulated by androgen while others were essentially unaffected. Subsequent pharmacological perturbation of the cellular process for energy generation confirmed that androgen starvation had a profound effect on this pathway. Conclusions: Our results provide evidence for the role of androgenic hormones in coordinating the expression of critical components involved in distinct cellular processes and further establish a foundation for the comprehensive reconstruction of androgen-regulated protein networks and pathways in prostate cancer cells.

Initial Proteome Analysis of Model Microorganism Haemophilus influenzae Strain Rd KW20

Abstract: The proteome of Haemophilus influenzae strain Rd KW20 was analyzed by liquid chromatography (LC) coupled with ion trap tandem mass spectrometry (MS/MS). This approach does not require a gel electrophoresis step and provides a rapidly developed snapshot of the proteome. In order to gain insight into the central metabolism of H. influenzae, cells were grown microaerobically and anaerobically in a rich medium and soluble and membrane proteins of strain Rd KW20 were proteolyzed with trypsin and directly examined by LC-MS/MS. Several different experimental and computational approaches were utilized to optimize the proteome coverage and to ensure statistically valid protein identification. Approximately 25% of all predicted proteins (open reading frames) of H. influenzae strain Rd KW20 were identified with high confidence, as their component peptides were unambiguously assigned to tandem mass spectra. Approximately 80% of the predicted ribosomal proteins were identified with high confidence, compared to the 33% of the predicted ribosomal proteins detected by previous two-dimensional gel electrophoresis studies. The results obtained in this study are generally consistent with those obtained from computational genome analysis, two-dimensional gel electrophoresis, and whole-genome transposon mutagenesis studies. At least 15 genes originally annotated as conserved hypothetical were found to encode expressed proteins. Two more proteins, previously annotated as predicted coding regions, were detected with high confidence; these proteins also have close homologs in related bacteria. The direct proteomics approach to studying protein expression in vivo reported here is a powerful method that is applicable to proteome analysis of any (micro)organism.

新软件工具在同位素编码亲和标签（ICAT）和串联质谱中的定量m88下载app分析中的应用：I。通过应用Icat和串联质谱法鉴定的肽序列和m88下载app的统计数据集与与T细胞共用的m88下载appLipid Rafts * s

Abstract: 根据来自Jurkat T细胞的标准方案通过T细胞受体/ CD28交联和来自对照（未刺激）细胞来制备脂质筏。来自对照和刺激的细胞制剂的共同分离m88下载app分别用同位素编码的亲和标签（ICAT）试剂的同位素正常（D0）和重（D8）和重（D8）型号标记。将样品合并，蛋白水解和通过阳离子交换色谱分级分级的结果肽。通过抗生物素蛋白 - 亲和色谱法通过ICAT试剂的生物素标签组分回收半胱氨酸（标记标记的）肽。在线微毛细管液相色谱串联质谱法在抗生物素素 - 亲和（ICAT标记）和流通（未标记）级分中进行。初始肽序列鉴定是通过使用Sequest™软件搜索记录的串联质谱法，用于使用Sequest™软件进行人的序列数据库。然后将新的统计数据建模算法应用于Sequest™搜索结果。这些可能在可能之间歧视"correct" and "incorrect"肽分配，以及它们通过计算它们的估计概率来分配它们的推断m88下载app，即每个肽分配和随后的m88下载app识别是"correct"人口。为方便起见，分配的肽序列的所得列表和它们对应的m88下载app在0.5的任意设定的截止截止时过滤。i.e. 50% likely to be "correct"）和上面并编译成两个单独的数据集。总共，这些数据集包含了7667个单独的肽鉴定，其代表了2669个独特的肽序列，对应于685个m88下载app和相关m88下载app基团。