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1.
With advances in ionization methods and instrumentation, liquid chromatography (LC)/mass spectrometry (MS) has become a powerful technology for protein characterization. This review article will describe the general approaches on LC-MS analysis in protein characterization, including bottom-up and top-down strategies. Discussions will be given on characterization of recombinant proteins, and post-translational and protein modifications such as disulfide bonds, glycosylation and phosphorylation using LC-MS. New research directions in this area will also be presented to illustrate future prospects of LC-MS in protein characterization, including application to proteomics.  相似文献   

2.
With advances in ionization methods and instrumentation, liquid chromatography (LC)/mass spectrometry (MS) has become a powerful technology for protein characterization. This review article will describe the general approaches on LC-MS analysis in protein characterization, including bottom-up and top-down strategies. Discussions will be given on characterization of recombinant proteins, and post-translational and protein modifications such as disulfide bonds, glycosylation and phosphorylation using LC-MS. New research directions in this area will also be presented to illustrate future prospects of LC-MS in protein characterization, including application to proteomics.  相似文献   

3.
Chemoproteomics represents a new research discipline at the interface of medicinal chemistry, biochemistry, and cell biology focused on studying the molecular mechanisms of action of drugs and other bioactive small molecules. Research strategies frequently combine phenotypic screening with subsequent target identification, and aim at a proteome-wide characterization of drug-induced changes in cellular protein expression and post-translational modifications. In recent years quantitative mass spectrometry has taken center stage in many of these approaches. This review describes experimental strategies in current chemical proteomics research, discusses recent examples of successful applications, and highlights areas in drug discovery where chemical proteomics-based assays using native endogenous proteins are expected to have substantial impact.  相似文献   

4.
This review outlines the concept of population proteomics and its implication in the discovery and validation of cancer-specific protein modulations. Population proteomics is an applied subdiscipline of proteomics engaging in the investigation of human proteins across and within populations to define and better understand protein diversity. Population proteomics focuses on interrogation of specific proteins from large number of individuals, utilizing top-down, targeted affinity mass spectrometry approaches to probe protein modifications. Deglycosylation, sequence truncations, side-chain residue modifications, and other modifications have been reported for myriad of proteins, yet little is know about their incidence rate in the general population. Such information can be gathered via population proteomics and would greatly aid the biomarker discovery efforts. Discovery of novel protein modifications is also expected from such large scale population proteomics, expanding the protein knowledge database. In regard to cancer protein biomarkers, their validation via population proteomics-based approaches is advantageous as mass spectrometry detection is used both in the discovery and validation process, which is essential for the detection of those structurally modified protein biomarkers.  相似文献   

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Mapping protein post-translational modifications with mass spectrometry   总被引:1,自引:0,他引:1  
Witze ES  Old WM  Resing KA  Ahn NG 《Nature methods》2007,4(10):798-806
Post-translational modifications of proteins control many biological processes, and examining their diversity is critical for understanding mechanisms of cell regulation. Mass spectrometry is a fundamental tool for detecting and mapping covalent modifications and quantifying their changes. Modern approaches have made large-scale experiments possible, screening complex mixtures of proteins for alterations in chemical modifications. By profiling protein chemistries, biologists can gain deeper insight into biological control. The aim of this review is introduce biologists to current strategies in mass spectrometry-based proteomics that are used to characterize protein post-translational modifications, noting strengths and shortcomings of various approaches.  相似文献   

8.
Electron capture dissociation (ECD) is a new fragmentation technique used in Fourier transform ion cyclotron resonance mass spectrometry and is complementary to traditional tandem mass spectrometry techniques. Disulfide bonds, normally stable to vibrational excitation, are preferentially cleaved in ECD. Fragmentation is fast and specific and labile post-translational modifications and non-covalent bonds often remain intact after backbone bond dissociation. ECD provides more extensive sequence coverage in polypeptides, and at higher electron energies even isoleucine and leucine are distinguishable. In biotechnology, the main area of ECD application is expected to be the top-down verification of DNA-predicted protein sequences, de novo sequencing, disulfide bond analysis and the combined top-down/bottom-up analysis of post-translational modifications.  相似文献   

9.
Electron transfer dissociation (ETD) has been developed recently as an efficient ion fragmentation technique in mass spectrometry (MS), being presently considered a step forward in proteomics with real perspectives for improvement, upgrade and application. Available also on affordable ion trap mass spectrometers, ETD induces specific N–Cα bond cleavages of the peptide backbone with the preservation of the post-translational modifications and generation of product ions that are diagnostic for the modification site(s). In addition, in the last few years ETD contributed significantly to the development of top-down approaches which enable tandem MS of intact protein ions. The present review, covering the last 5 years highlights concisely the major achievements and the current applications of ETD fragmentation technique in proteomics. An ample part of the review is dedicated to ETD contribution in the elucidation of the most common posttranslational modifications, such as phosphorylation and glycosylation. Further, a brief section is devoted to top-down by ETD method applied to intact proteins. As the last few years have witnessed a major expansion of the microfluidics systems, a few considerations on ETD in combination with chip-based nanoelectrospray (nanoESI) as a platform for high throughput top-down proteomics are also presented.  相似文献   

10.
高分辨率质谱技术的快速发展使得"自顶向下"的蛋白质组学(top-down proteomics)研究逐渐成熟起来.在完整蛋白质水平上研究蛋白质组可以提供更精准、更丰富的生物学信息,特别是对于蛋白质上发生了多种关联性的翻译后修饰的情况.另外,由于基因突变、RNA可变剪接和大量蛋白质翻译后修饰的存在,同一个基因往往最终会产生多个"蛋白质变体"(proteoform),而要准确地鉴定这些蛋白质变体,也离不开"自顶向下"的蛋白质组学.在蛋白质水平上的分离技术、质谱技术与生物信息学技术是完整蛋白质鉴定最关键的三项技术.高效的分离技术是实现规模化蛋白质变体鉴定的前提,有效的质谱碎裂是提供可靠鉴定的核心,而快速准确的质谱鉴定算法则是数据分析效率的保障.本文对这三项技术进行了详细总结,重点集中在生物信息学相关技术上,包括对完整蛋白质的质谱数据预处理、数据库搜索鉴定以及翻译后修饰定位等几个计算问题的讨论.  相似文献   

11.
Today, proteomics usually compares clinical samples by use of bottom-up profiling with high resolution mass spectrometry, where all protein products of a single gene are considered as an integral whole. At the same time, proteomics of proteoforms, which considers the variety of protein species, offers the potential to discover valuable biomarkers. Proteoforms are protein species that arise as a consequence of genetic polymorphisms, alternative splicing, post-translational modifications and other less-explored molecular events. The comprehensive observation of proteoforms has been an exclusive privilege of top-down proteomics. Here, we review the possibilities of a bottom-up approach to address the microheterogeneity of the human proteome. Special focus is given to shotgun proteomics and structure-based bioinformatics as a source of hypothetical proteoforms, which can potentially be verified by targeted mass spectrometry to determine the relevance of proteoforms to diseases.  相似文献   

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James DC 《Cytotechnology》1996,22(1-3):17-24
The advent of new technologies for analysis of biopolymers by mass spectrometry has revolutionised strategies for recombinant protein characterization. The principal recent developments have been matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry. Using these tools, accurate molecular mass determinations can now be obtained routinely-often using minute (picomole-femtomole) quantities of protein or protein fragments. These techniques have proved indispensible for detailed characterization of the post-translational modifications of recombinant proteins produced by eukaryotic systems. Glycosylation is arguably the most important and complex of these modifications and has prompted widespread use of these new techniques. In this mini-review article I describe recent advances in the use of mass spectrometry for analysis of recombinant glycoproteins.  相似文献   

14.
Qin  Weiwei  Wang  Ting  Huang  He  Gao  Youhe 《中国科学:生命科学英文版》2019,62(11):1514-1520
A biomarker is a measurable indicator associated with changes in physiological state or disease. In contrast to the blood which is under homeostatic controls, urine reflects changes in the body earlier and more sensitively, and is therefore a better biomarker source. Lysine acetylation is an abundant and highly regulated post-translational modification. It plays a pivotal role in modulating diverse biological processes and is associated with various important diseases. Enrichment or visualization of proteins with specific post-translational modifications provides a method for sampling the urinary proteome and reducing sample complexity. In this study, we used anti-acetyllysine antibody-based immunoaffinity enrichment combined with high-resolution mass spectrometry to profile lysine-acetylated proteins in normal human urine. A total of 629 acetylation sites on 315 proteins were identified, including some very low-abundance proteins. This is the first proteome-wide characterization of lysine acetylation proteins in normal human urine. Our dataset provides a useful resource for the further discovery of lysine-acetylated proteins as biomarkers in urine.  相似文献   

15.
The diverse proteome of an organism arises from such events as single nucleotide substitutions at the DNA level, different RNA processing, and dynamic enzymatic post-translational modifications. This minireview focuses on the measurement of intact proteins to describe the diversity found in proteomes. The field of biological mass spectrometry has steadily advanced, enabling improvements in the characterization of single proteins to proteins derived from cells or tissues. In this minireview, we discuss the basic technology for "top-down" intact protein analysis. Furthermore, examples of studies involved with the qualitative and quantitative analysis of full-length polypeptides are provided.  相似文献   

16.
Zhang J  Zhang H  Ayaz-Guner S  Chen YC  Dong X  Xu Q  Ge Y 《Biochemistry》2011,50(27):6081-6092
Cardiac troponin T (cTnT), the tropomyosin binding subunit of the troponin complex, plays a pivotal regulatory role in the Ca(2+)-mediated interaction between actin thin filament and myosin thick filament. The post-translational modifications (PTMs) and alternative splicing of cTnT may represent important regulatory mechanisms of cardiac contractility. However, a complete characterization of PTMs and alternatively spliced isoforms in cTnT present in vivo is lacking. Top-down protein mass spectrometry (MS) analyzes whole proteins, thus providing a global view of all types of modifications, including PTMs and sequence variants, simultaneously in one spectrum without a priori knowledge. In this study, we applied an integrated immunoaffinity chromatography and top-down MS approach to comprehensively characterize PTMs and alternatively spliced isoforms of cTnT purified from healthy human and wild-type mouse heart tissue. High-resolution Fourier transform MS revealed that human cTnT (hcTnT) and mouse cTnT (mcTnT) have similar phosphorylation patterns, whereas higher molecular heterogeneity was observed for mcTnT than hcTnT. Further MS/MS fragmentation of monophosphorylated hcTnT and mcTnT by electron capture dissociation and collisionally activated dissociation unambiguously identified Ser1 as the conserved in vivo phosphorylation site. In contrast, we identified a single spliced isoform for hcTnT but three alternatively spliced isoforms for mcTnT. Moreover, we observed distinct proteolytic degradation products for hcTnT and mcTnT. This study also demonstrates the advantage of top-down MS/MS with complementary fragmentation techniques for the identification of modification sites in the highly acidic N-terminal region of cTnT.  相似文献   

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Here we present a comprehensive method for proteome analysis that integrates both intact protein measurement ("top-down") and proteolytic fragment characterization ("bottom-up") mass spectrometric approaches, capitalizing on the unique capabilities of each method. This integrated approach was applied in a preliminary proteomic analysis of Shewanella oneidensis, a metal-reducing microbe of potential importance to the field of bioremediation. Cellular lysates were examined directly by the "bottom-up" approach as well as fractionated via anion-exchange liquid chromatography for integrated studies. A portion of each fraction was proteolytically digested, with the resulting peptides characterized by on-line liquid chromatography/tandem mass spectrometry. The remaining portion of each fraction containing the intact proteins was examined by high-resolution Fourier transform mass spectrometry. This "top-down" technique provided direct measurement of the molecular masses for the intact proteins and thereby enabled confirmation of post-translational modifications, signal peptides, and gene start sites of proteins detected in the "bottom-up" experiments. A total of 868 proteins from virtually every functional class, including hypotheticals, were identified from this organism.  相似文献   

19.
Amide hydrogen/deuterium exchange detected by mass spectrometry (HXMS) is seeing wider use for the identification of intrinsically disordered parts of proteins. In this review, we discuss examples of how discovery of intrinsically disordered regions and their removal can aid in structure determination, biopharmaceutical quality control, the characterization of how post-translational modifications affect weak structuring of disordered regions, the study of coupled folding and binding, and the characterization of amyloid formation. This article is part of a Special Issue entitled: Mass spectrometry in structural biology.  相似文献   

20.
Fragmentation and "top-down" sequencing of intact proteins by mass spectrometry (MS) is most commonly performed by infusion of protein solutions into Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers. However, the high cost of this instrumentation, coupled with the need to infuse "clean" solutions (lacking standard biological buffers), limits broad application of this technique. The current study describes an alternative approach to top-down sequencing using in-source fragmentation on quadrupole time-of-flight (Q-Tof) instrumentation coupled with reversed-phase liquid chromatography (LC). Application of this technique to purified recombinant samples yielded protein fragments during routine LC-MS analysis. The presence of multiple N- and C-terminal fragments allowed localization of structural modifications without proteolytic digestion. The method was extended to complex samples by using LC conditions that provided high-resolution protein separation. Utility of the method was illustrated by real-time monitoring of protein modifications occurring in reconstituted apoptosomes. These experiments illustrate that intact protein mass and limited sequence information can be obtained simultaneously on an LC timescale. This approach will allow a wide variety of laboratories to routinely apply top-down sequencing to problems in structural characterization, protein purification, and biomarker identification.  相似文献   

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