膜整合蛋白质的“鸟枪法”蛋白质组学分析策略

疾病状态下生物膜表面蛋白质分子标记的表达量和修饰状态会发生改变。但由于其低丰度和不易溶解等特性,制约了膜蛋白质组学的研究,同时也制约了相关药物靶标的设计。近年来,为克服这些困难,学者们提出了"鸟枪法"的膜蛋白质组学研究策略。基于此,本文论述了"鸟枪法"的蛋白质组学分析的基本过程及其后续的部分改进工作。随着新的策略不断被采用,更多膜蛋白质的拓扑学特征和功能的相关研究一定会走上新的台阶。     

Characterization of the Theileria parva sporozoite proteome

East Coast fever is a lymphoproliferative disease caused by the tick-borne protozoan parasite Theileria parva. The sporozoite stage of this parasite, harboured and released from the salivary glands of the tick Rhipicephalus appendiculatus during feeding, invades and establishes infection in bovine lymphocytes. Blocking this initial stage of invasion presents a promising vaccine strategy for control of East Coast fever and can in part be achieved by targeting the major sporozoite surface protein p67. To support research on the biology of T. parva and the identification of additional candidate vaccine antigens, we report on the sporozoite proteome as defined by LC–MS/MS analysis. In total, 4780 proteins were identified in an enriched preparation of sporozoites. Of these, 2007 were identified as T. parva proteins, representing close to 50% of the total predicted parasite proteome. The remaining 2773 proteins were derived from the tick vector. The identified sporozoite proteins include a set of known T. parva antigens targeted by antibodies and cytotoxic T cells from cattle that are immune to East Coast fever. We also identified proteins predicted to be orthologs of Plasmodium falciparum sporozoite surface molecules and invasion organelle proteins, and proteins that may contribute to the phenomenon of bovine lymphocyte transformation. Overall, these data establish a protein expression profile of T. parva sporozoites as an important starting point for further study of a parasitic species which has considerable agricultural impact.     

The two‐phase partitioning system – a powerful technique to purify integral membrane proteins of Corynebacterium glutamicum for quantitative shotgun analysis

We established a single consecutive strategy which assigned the most comprehensive number of integral membrane proteins from Gram‐positive bacteria to date. For this purpose, we adapted a biphasic partitioning system for the biotechnologically intensively used Corynebacterium glutamicum and proved for the first time that such a system is well suited for quantitative comparison. 297 integral membrane proteins were identified by our integrated approach, which depletes stringently cytosolic proteins. In combination with our previously developed SIMPLE strategy, our data comprise 61% (374 integral membrane proteins) of the entire membrane proteome, which aims towards an almost comprehensive coverage. Wild type and a production strain of C. glutamicum were compared by ¹⁵N metabolic labelling and quantitation was obtained by spectral counting and peak areas. Both quantification strategies display a consistent trend in up or downregulation of proteins. Nevertheless, spectral counting often provides results indicating a much stronger regulation compared to ProRata values. Either spectral counting seems to exaggerate protein regulation or ProRata tends to attenuate the information about the regulation level. We highlight some of the biologically relevant candidates, which prove that our approach helps to give a deeper quantitative insight towards the understanding of transport and other membrane associated processes, important for strain development of C. glutamicum.     

The cancer cell secretome: A good source for discovering biomarkers?

Cancer is a leading cause of death. Early detection is usually associated with better clinical outcomes. Recent advances in genomics and proteomics raised hopes that new biomarkers for diagnosis, prognosis or monitoring therapeutic response will soon be discovered. Proteins secreted by cancer cells, referred also as “the cancer cell secretome”, is a promising source for biomarker discovery. In this review we will summarize recent advances in cancer cell secretome analysis, focusing on the five most fatal cancers (lung, breast, prostate, colorectal, and pancreatic). For each cancer type we will describe the proteomic approaches utilized for the identification of novel biomarkers. Despite progress, identification of markers that are superior to those currently used has proven to be a difficult task and very few, if any, newly discovered biomarker has entered the clinic the last 10 years.     

Analyzing chromatin remodeling complexes using shotgun proteomics and normalized spectral abundance factors

Mass spectrometry-based approaches are commonly used to identify proteins from multiprotein complexes, typically with the goal of identifying new complex members or identifying post-translational modifications. However, with the recent demonstration that spectral counting is a powerful quantitative proteomic approach, the analysis of multiprotein complexes by mass spectrometry can be reconsidered in certain cases. Using the chromatography-based approach named multidimensional protein identification technology, multiprotein complexes may be analyzed quantitatively using the normalized spectral abundance factor that allows comparison of multiple independent analyses of samples. This study describes an approach to visualize multiprotein complex datasets that provides structure function information that is superior to tabular lists of data. In this method review, we describe a reanalysis of the Rpd3/Sin3 small and large histone deacetylase complexes previously described in a tabular form to demonstrate the normalized spectral abundance factor approach.     

Proteomic Analysis to Identify Tightly-Bound Cell Wall Protein in Rice Calli

Rice is a model plant widely used for basic and applied research programs. Plant cell wall proteins play key roles in a broad range of biological processes. However, presently, knowledge on the rice cell wall proteome is rudimentary in nature. In the present study, the tightly-bound cell wall proteome of rice callus cultured cells using sequential extraction protocols was developed using mass spectrometry and bioinformatics methods, leading to the identification of 1568 candidate proteins. Based on bioinformatics analyses, 389 classical rice cell wall proteins, possessing a signal peptide, and 334 putative non-classical cell wall proteins, lacking a signal peptide, were identified. By combining previously established rice cell wall protein databases with current data for the classical rice cell wall proteins, a comprehensive rice cell wall proteome, comprised of 496 proteins, was constructed. A comparative analysis of the rice and Arabidopsis cell wall proteomes revealed a high level of homology, suggesting a predominant conservation between monocot and eudicot cell wall proteins. This study importantly increased information on cell wall proteins, which serves for future functional analyses of these identified rice cell wall proteins.     

Pupylated proteins in Corynebacterium glutamicum revealed by MudPIT analysis

In a manner similar to ubiquitin, the prokaryotic ubiquitin‐like protein (Pup) has been shown to target proteins for degradation via the proteasome in mycobacteria. However, not all actinobacteria possessing the Pup protein also contain a proteasome. In this study, we set out to study pupylation in the proteasome‐lacking non‐pathogenic model organism Corynebacterium glutamicum. A defined pup deletion mutant of C. glutamicum ATCC 13032 grew aerobically as the parent strain in standard glucose minimal medium, indicating that pupylation is dispensable under these conditions. After expression of a Pup derivative carrying an aminoterminal polyhistidine tag in the Δpup mutant and Ni²⁺‐chelate affinity chromatography, pupylated proteins were isolated. Multidimensional protein identification technology (MudPIT) and MALDI‐TOF‐MS/MS of the elution fraction unraveled 55 proteins being pupylated in C. glutamicum and 66 pupylation sites. Similar to mycobacteria, the majority of pupylated proteins are involved in metabolism or translation. Our results define the first pupylome of an actinobacterial species lacking a proteasome, confirming that other fates besides proteasomal degradation are possible for pupylated proteins.     

Shotgun proteomic analysis for detecting differentially expressed proteins in the reduced culm number rice

To survey protein expression patterns in the reduced culm number (RCN) rice, a comparative shotgun proteomic analysis was conducted. For large-scale protein identification, multidimensional protein identification technology (MudPIT) coupled with pre-fractionation of plant shoot proteins led to the identification of 3004 non-redundant rice proteins. By statistically comparing relative amounts of 1353 reproducibly identified proteins between the RCN rice and the wild-type rice, 44 differentially expressed proteins were detected, where 42 proteins were increased and 2 proteins were decreased in the RCN rice. These proteins appear to have roles in glycolysis, trichloroacetic acid cycle, secondary metabolism, nutrient recycling, and nucleotide metabolism and repair. Consequently, we hypothesized that the RCN rice might fail to maintain sugar nutrient homeostasis. This was confirmed with the observation that the sucrose concentration was increased significantly in the RCN rice compared with the wild-type rice. Also, the RCN rice showed a hypersensitive response to exogenous sucrose treatment.