Recent progress in liquid chromatography-based separation and label-free quantitative plant proteomics

Recent innovations in liquid chromatography-mass spectrometry (LC-MS)-based methods have facilitated quantitative and functional proteomic analyses of large numbers of proteins derived from complex samples without any need for protein or peptide labelling. Regardless of its great potential, the application of these proteomics techniques to plant science started only recently. Here we present an overview of label-free quantitative proteomics features and their employment for analysing plants. Recent methods used for quantitative protein analyses by MS techniques are summarized and major challenges associated with label-free LC-MS-based approaches, including sample preparation, peptide separation, quantification and kinetic studies, are discussed. Database search algorithms and specific aspects regarding protein identification of non-sequenced organisms are also addressed. So far, label-free LC-MS in plant science has been used to establish cellular or subcellular proteome maps, characterize plant-pathogen interactions or stress defence reactions, and for profiling protein patterns during developmental processes. Improvements in both, analytical platforms (separation technology and bioinformatics/statistical analysis) and high throughput nucleotide sequencing technologies will enhance the power of this method.     

Virus-induced gene silencing in detached tomatoes and biochemical effects of phytoene desaturase gene silencing

Virus-induced gene silencing (VIGS) is a technology that has rapidly emerged for gene function studies in plants. Many advances have been made in applying this technique in an increasing number of crops. Recently, VIGS has been successfully used to silence genes in tomato fruit through agroinfiltration of fruit attached to the plant. The phytoene desaturase (Pds) gene has been widely used as a reporter gene in VIGS experiments, although little is known about the changes that occur due to its silencing in plants. In this paper, we describe the efficient silencing of the Pds gene through the VIGS approach in detached tomato fruits, which makes the VIGS procedure even more versatile and applicable. After 16 days of agroinfiltration, approximately 75% of the tomatoes showed Pds silencing symptoms, although the distribution of silenced areas was variable among fruits. To study the potential effects caused by Pds silencing in detached tomatoes, carotenoids and other semi-polar secondary metabolites were analyzed using Liquid Chromatography-Mass Spectrometry. In addition, potential differences in primary metabolites were analyzed using Gas Chromatography-Mass Spectrometry. The results indicated that the yellow phenotype observed in Pds-silenced fruit was mainly due to the lack of the red-colored lycopene and therefore to a more pronounced contribution of the yellow-orange carotenoids (lutein, violaxanthin, and zeaxanthin) to the final color of the fruits. Furthermore, the biochemical changes observed in Pds-silenced detached tomatoes suggested that carotenoid and other pathways, e.g. leading to alkaloids and flavonoids, might be affected by the silencing of this reporter gene, and this should be taken into consideration for future experimental designs.     

Identification of novel oxidized levuglandin D2 in marine red alga and mouse tissue

In animals, the product of cyclooxygenase reacting with arachidonic acid, prostaglandin(PG)H(2), can undergo spontaneous rearrangement and nonenzymatic ring cleavage to form levuglandin(LG)E(2) and LGD(2). These LGs and their isomers are highly reactive γ-ketoaldehydes that form covalent adducts with proteins, DNA, and phosphatidylethanolamine in cells. Here, we isolated a novel oxidized LGD(2) (ox-LGD(2)) from the red alga Gracilaria edulis and determined its planar structure. Additionally, ox-LGD(2) was identified in some tissues of mice and in the lysate of phorbol-12-myristate-13-acetate (PMA)-treated THP-1 cells incubated with arachidonic acid using LC-MS/MS. These results suggest that ox-LGD(2) is a common oxidized metabolite of LGD(2). In the planar structure of ox-LGD(2), H8 and H12 of LGD(2) were dehydrogenated and the C9 aldehyde was oxidized to a carboxylic acid, which formed a lactone ring with the hydrated ketone at C11. These structural differences imply that ox-LGD(2) is less reactive with amines than LGs. Therefore, ox-LGD(2) might be considered a detoxification metabolite of LGD(2).     

Histone H3 lysine 27 acetylation is altered in colon cancer

Background

Histone post-translational modifications (PTMs) play an important role in the regulation of the expression of genes, including those involved in cancer development and progression. However, our knowledge of PTM patterns in human tumours is limited.

Methods

MS-based analyses were used to quantify global alterations of histone PTMs in colorectal cancer (CRC) samples. Histones isolated from 12 CRCs and their corresponding normal mucosa by acidic extraction were separated by SDS-PAGE and analysed by liquid chromatography-mass spectrometry.

Results

Among 96 modified peptides, 41 distinct PTM sites were identified, of which 7, 13, 11, and 10 were located within the H2A, H2B, H3, and H4 sequences, respectively, and distributed among the amino-terminal tails and the globular domain of the four histones. Modification intensities were quantified for 33 sites, of which 4 showed significant (p-value ≤ 0.05) differences between CRC tissues and healthy mucosa samples. We identified histone H3 lysine 27 acetylation (H3K27Ac) as a modification upregulated in CRC, which had not been shown previously.

Conclusions

The present results indicate the usefulness of a bottom-up proteomic approach for the detection of histone modifications at a global scale. The differential abundance of H3K27Ac mark in CRC, a PTM associated with active enhancers, suggests its role in regulating genes whose expression changes in CRC.     

Rapid development of proteomics in China: from the perspective of the Human Liver Proteome Project and technology development

Proteomics focuses on the systematic identification and quantification of entire proteomes and interpretation of proteins’biological functions.During the last decade,proteomics in China has grown much faster than other research fields in life sciences.At the beginning of the second decade of the 21st century,the rapid development of high-resolution and high-speed mass spectrometry makes proteomics a powerful tool to study the mechanisms underlying physiological/pathological processes in organisms.This article provides a brief overview of proteomics technology development and representative scientific progress of the Human Liver Proteome Project in China over the past three years.     

红凤菜新鲜茎叶中总黄酮提取物的LC-MS分析

红凤菜（Gynura bicolor DC.）为菊科（Compositae）菊三七属（Gynura Cass.）植物,别名血皮菜、观音菜、观音苋、紫背天葵等,主要分布于中国南方各地,全草均可入药[1];也可作为蔬菜食用,在国内多个地区均有栽培和销售。目前已知红凤菜含有黄酮类、酚性酸类、萜类、甾醇类、脂肪酸类、生物碱类及花青素类等[2-7]成分。由于黄酮类成分多具有保护心血管、抗肿瘤、抗糖尿病、抗氧化、抗炎和抗病毒等作用[8],因此对红凤菜中黄酮类成分的研究具有深度开发价值。     

雷诺嗪缓释片在比格犬体内的药代动力学

目的:研究雷诺嗪缓释片在比格犬体内的药物代谢动力学,并与参照制剂比较,为其是否具有缓释特征提供依据。方法:首先建立血浆中雷诺嗪浓度的液相色谱-串联质谱联用检测方法,并考察方法的专属性、准确度、日内日间精密度、回收率、线性范围等。采用随机对照试验设计,将12只比格犬随机分为A、B组,每组6只,分别服用1片雷诺嗪缓释片(500 mg/片)和1片参比制剂雷诺嗪片(500 mg/片),均于给药前和给药后不同时间点采集血样,用已建立的液质联用方法检测血样中雷诺嗪的血药浓度,计算2组比格犬的药代动力学参数。结果:受试组和参照组半衰期t1/2分别为13.3±8.3和2.36±0.92 h,峰浓度Cmax分别为923.9±340.5和3205±1314 ng/mL,达峰时间Tmax分别为1.6±0.38和0.88±0.14 h,曲线下面积AUC0~∞分别为6252.1±2860.3和9916±4305(ng·h)/mL,清除率Cl分别为11.3±9.8和6.39±3.95 L/(kg·h)。受试制剂雷诺嗪缓释片和参比制剂雷诺嗪片的药代特征和血药浓度-时间变化趋势明显不同,受试组血药浓度缓慢上升和下降,峰值较低;而参照组血药浓度峰值显著高于受试组,有明显的突释效应。结论:液质联用检测方法准确可靠,适合体内药代动力学研究;与参比制剂雷诺嗪片相比,受试制剂雷诺嗪缓释片符合缓释片的基本药代动力学特点。     

High-throughput glycosylation analysis of therapeutic immunoglobulin G by capillary gel electrophoresis using a DNA analyzer

The Fc glycosylation of therapeutic antibodies is crucial for their effector functions and their behavior in pharmacokinetics and pharmacodynamics. To monitor the Fc glycosylation in bioprocess development and characterization, high-throughput techniques for glycosylation analysis are needed. Here, we describe the development of a largely automated high-throughput glycosylation profiling method with multiplexing capillary-gel-electrophoresis (CGE) with laser induced fluorescence (LIF) detection using a DNA analyzer. After PNGaseF digestion, the released glycans were labeled with 9-aminopyrene-1,3,6-trisulfonic acid (APTS) in 96-well plates, which was followed by the simultaneous analysis of up to 48 samples. The peak assignment was conducted by HILIC-UPLC-MS/MS of the APTS-labeled glycans combined with peak fractionation and subsequent CGE-LIF analysis of the MS-characterized fractions. Quantitative data evaluation of the various IgG glycans was performed automatically using an in-house developed software solution. The excellent method accuracy and repeatability of the test system was verified by comparison with two UPLC-based methods for glycan analysis. Finally, the practical value of the developed method was demonstrated by analyzing the antibody glycosylation profiles from fermentation broths after small scale protein A purification.     

Artifactual FA dimers mimic FAHFA signals in untargeted metabolomics pipelines

FA esters of hydroxy FAs (FAHFAs) are lipokines with extensive structural and regional isomeric diversity that impact multiple physiological functions, including insulin sensitivity and glucose homeostasis. Because of their low molar abundance, FAHFAs are typically quantified using highly sensitive LC-MS/MS methods. Numerous relevant MS databases house in silico-spectra that allow identification and speciation of FAHFAs. These provisional chemical feature assignments provide a useful starting point but could lead to misidentification. To address this possibility, we analyzed human serum with a commonly applied high-resolution LC-MS untargeted metabolomics platform. We found that many chemical features are putatively assigned to the FAHFA lipid class based on exact mass and fragmentation patterns matching spectral databases. Careful validation using authentic standards revealed that many investigated signals provisionally assigned as FAHFAs are in fact FA dimers formed in the LC-MS pipeline. These isobaric FA dimers differ structurally only by the presence of an olefinic bond. Furthermore, stable isotope-labeled oleic acid spiked into human serum at subphysiological concentrations showed concentration-dependent formation of a diverse repertoire of FA dimers that analytically mimicked FAHFAs. Conversely, validated FAHFA species did not form spontaneously in the LC-MS pipeline. Together, these findings underscore that FAHFAs are endogenous lipid species.  However, nonbiological FA dimers forming in the setting of high concentrations of FFAs can be misidentified as FAHFAs. Based on these results, we assembled a FA dimer database to identify nonbiological FA dimers in untargeted metabolomics datasets.     

Characterization of Glycoproteoforms of Integrins α2 and β1 in Megakaryocytes in the Occurrence of JAK2V617F Mutation-Induced Primary Myelofibrosis

Primary myelofibrosis (PMF) is a neoplasm prone to leukemic transformation, for which limited treatment is available. Among individuals diagnosed with PMF, the most prevalent mutation is the JAK2V617F somatic point mutation that activates the Janus kinase 2 (JAK2) enzyme. Our earlier reports on hyperactivity of β1 integrin and enhanced adhesion activity of the α2β1 complex in JAK2V617F megakaryocytes (MKs) led us to examine the new hypothesis that this mutation leads to posttranslational modification via changes in glycosylation. Samples were derived from immunoprecipitation of MKs obtained from Vav1-hJAK2^V617F and WT mice. Immunoprecipitated fractions were separated by SDS-PAGE and analyzed using LC-MS/MS techniques in a bottom-up glycoproteomics workflow. In the immunoprecipitate, glycopeptiforms corresponding to 11 out of the 12 potential N-glycosylation sites of integrin β1 and to all nine potential glycosylation sites of integrin α2 were observed. Glycopeptiforms were compared across WT and JAK2V617F phenotypes for both integrins. The overall trend observed is that JAK2V617F mutation in PMF MKs leads to changes in β1 glycosylation; in most cases, it results in an increase in the integrated area of glycopeptiforms. We also observed that in mutated MKs, changes in integrin α2 glycosylation were more substantial than those observed for integrin β1 glycosylation, a finding that suggests that altered integrin α2 glycosylation may also affect activation. Additionally, the identification of proteins associated to the cytoskeleton that were co-immunoprecipitated with integrins α2 and β1 demonstrated the potential of the methodology employed in this study to provide some insight, at the peptide level, into the consequences of integrin activation in MKs. The extensive and detailed glycosylation patterns we uncovered provide a basis for future functional studies of each site in control cells as compared to JAK2V617F-mutated cells. Data are available via ProteomeXchange with identifier PXD030550.