Efficient Enrichment Method Research Articles

Specific enrichment of a targeted nitrotyrosine-containing peptide from complex matrices and relative quantification for liquid chromatography–mass spectrometry analysis

Protein tyrosine nitration is considered an important non-enzymatic post-translational modification. In the tyrosine nitration process, 3-nitrotyrosine is formed and recognized as a biomarker of nitrosative/nitrative stress implicated in inflammatory responses and age-related disorders. In view of the complexity of biological samples and the ultra-low abundance of protein-incorporated nitrotyrosine, selective enrichment of nitrotyrosine-containing peptides prior to chromatographic separation is crucial. Herein, I report a simple yet highly specific and efficient enrichment method for nitrotyrosine-containing peptides. After blocking all primary amines in the sample by acetylation with acetic anhydride, I then further converted all nitrotyrosine residues into aminotyrosine residues by reduction with dithiothreitol and hemin. Therefore, I eliminated the side-product with 80Da adduct, since inevitable considerable amount of which was generated in the widely used reduction mediated by sodium dithionite. Both acetylation and reduction yields were close to 100%, and my one-pot sample derivatization applied no solid phase extraction steps or sample transference to avoid sample loss. To capture and release aminotyrosine-containing peptides, I synthesized an N-hydroxysuccinimide-ester-functionalized stationary phase which had very high affinity towards amino groups and possessed a base-cleavable ester linker to retrieve targeted peptides by hydrolysis. I validated this strategy by highly efficient enrichment of the targeted peptide from complex matrices of trypsin-digested bovine serum albumin (BSA) and human plasma spiked with derivatized nitrotyrosine-containing angiotensin II. My enrichment method successfully removed most untargeted peptides in those samples. By relative quantification with home-made identical and stable-isotope labelled internal standards, I investigated the recoveries of a nitrotyrosine-containing peptide from complex biological matrices during enrichment for the first time. Mean recoveries were 49.8% and 41.1% (n=6) for the enrichment of nitrotyrosine-containing angiotensin II from 1:100 (w/w) BSA digest and from 1:10 000 (w/w) human plasma digest, respectively. My enrichment method demonstrated great potential in future applications to clinical samples and biomarker discovery.

Computational and Statistical Methods for High-Throughput Mass Spectrometry-Based PTM Analysis.

Cell signaling and functions heavily rely on post-translational modifications (PTMs) of proteins. Their high-throughput characterization is thus of utmost interest for multiple biological and medical investigations. In combination with efficient enrichment methods, peptide mass spectrometry analysis allows the quantitative comparison of thousands of modified peptides over different conditions. However, the large and complex datasets produced pose multiple data interpretation challenges, ranging from spectral interpretation to statistical and multivariate analyses. Here, we present a typical workflow to interpret such data.

Highly Reproducible Ag NPs/CNT-Intercalated GO Membranes for Enrichment and SERS Detection of Antibiotics.

The increasing pollution of aquatic environments by antibiotics makes it necessary to develop efficient enrichment and sensitive detection methods for environmental antibiotics monitoring. In this work, silver nanoparticles and carbon nanotube-intercalated graphene oxide laminar membranes (Ag NPs/CNT-GO membranes) were successfully prepared for enrichment and surface-enhanced Raman scattering (SERS) detection of antibiotics. The prepared Ag NPs/CNT-GO membranes exhibited a high enrichment ability because of the π-π stacking and electrostatic interactions of GO toward antibiotic molecules, which enhanced the sensitivity of SERS measurements and enabled the antibiotics to be determined at sub-nM concentrations. In addition, the nanochannels created by the intercalation of CNTs into GO layers resulted in an 8-fold enhancement in the water permeance of Ag NPs/CNT-GO membranes compared to that of pure GO membranes. More importantly, the Ag NPs/CNT-GO membranes exhibited high reproducibility and long-term stability. The spot-to-spot variation in SERS intensity was less than 15%, and the SERS performance was maintained for at least 70 days. The Ag NPs/CNT-GO membranes were also used for SERS detection of antibiotics in real samples; the results showed that the characteristic peaks of antibiotics were obviously recognizable. Thus, the sensitive SERS detection of antibiotics based on Ag NPs/CNT-GO offers great potential for practical applications in environmental analysis.

Efficient enrichment of glycopeptides with sulfonic acid-functionalized mesoporous silica

This work presents an efficient and selective enrichment method for glycoprotein/glycopeptides with sulfonic acid-functionalized mesoporous silica (SBA-15-SO3H), which is synthesized via simple oxidation of -SH groups with H2O2. The functionalized SBA-15 shows large surface area and accessible pores, and can selectively adsorb glycopeptides via hydrogen bond and hydrophilic interaction. Upon the selective enrichment prior to the mass spectrometric (MS) analysis, the signals of glycopeptides are significantly enhanced, which leads to the identifiable signals of 21 glycopeptides from the digest of HRP, 16 glycopeptides from the digest of human IgG, and 16 glycopeptides from the digest of chicken avidin. The SBA-15-SO3H gives significant selectivity for glycopeptides even at a low molar ratio of glycopeptides to nonglycopeptides with an enrichment time of 15min. Therefore, this work provides a powerful material for selective enrichment and identification of low abundant glycopeptides in glycoproteomic analysis.

A Novel Quantitative Mass Spectrometry Platform for Determining Protein O-GlcNAcylation Dynamics

Over the past decades, protein O-GlcNAcylation has been found to play a fundamental role in cell cycle control, metabolism, transcriptional regulation, and cellular signaling. Nevertheless, quantitative approaches to determine in vivo GlcNAc dynamics at a large-scale are still not readily available. Here, we have developed an approach to isotopically label O-GlcNAc modifications on proteins by producing (13)C-labeled UDP-GlcNAc from (13)C6-glucose via the hexosamine biosynthetic pathway. This metabolic labeling was combined with quantitative mass spectrometry-based proteomics to determine protein O-GlcNAcylation turnover rates. First, an efficient enrichment method for O-GlcNAc peptides was developed with the use of phenylboronic acid solid-phase extraction and anhydrous DMSO. The near stoichiometry reaction between the diol of GlcNAc and boronic acid dramatically improved the enrichment efficiency. Additionally, our kinetic model for turnover rates integrates both metabolomic and proteomic data, which increase the accuracy of the turnover rate estimation. Other advantages of this metabolic labeling method include in vivo application, direct labeling of the O-GlcNAc sites and higher confidence for site identification. Concentrating only on nuclear localized GlcNAc modified proteins, we are able to identify 105 O-GlcNAc peptides on 42 proteins and determine turnover rates of 20 O-GlcNAc peptides from 14 proteins extracted from HeLa nuclei. In general, we found O-GlcNAcylation turnover rates are slower than those published for phosphorylation or acetylation. Nevertheless, the rates widely varied depending on both the protein and the residue modified. We believe this methodology can be broadly applied to reveal turnovers/dynamics of protein O-GlcNAcylation from different biological states and will provide more information on the significance of O-GlcNAcylation, enabling us to study the temporal dynamics of this critical modification for the first time.

Conjugated Microporous Polymers with Built-In Magnetic Nanoparticles for Excellent Enrichment of Trace Hydroxylated Polycyclic Aromatic Hydrocarbons in Human Urine.

Conjugated microporous polymers (CMPs), linked by a covalent bond to form an extension of the aromatic ring skeleton, are microporous materials characterized by a highly conjugated structure and high stability. The present study reported on a novel strategy for the synthesis of CMPs with built-in magnetic nanoparticles for excellent enrichment of trace hydroxylated polycyclic aromatic hydrocarbons (OH-PAHs) in human urine. We modified Fe3O4 nanoparticles with boronic acid groups and then reacted the nanoparticles with reactive monomers of polyphenylene conjugated microporous polymer (PP-CMP) to anchor the magnetic components in the PP-CMP framework. Chemical bonding between Fe3O4 nanoparticles and PP-CMP networks, together with equally firm covalent linkage and rigidity of the PP-CMP network, endows the magnetic PP-CMP with remarkable chemical stability and durability, even in harsh conditions. Magnetic PP-CMP has the characteristics of high conjugation ability, highly porous structure, and magnetism, which makes it an ideal magnetic adsorbent for trace analytes with aromatic conjugation structure. The adsorption mechanism of OH-PAHs on magnetic PP-CMP was investigated and demonstrated that hydrophobic interaction was important for the contribution of interaction between adsorbents and target analytes, together with the assistance of π-π stacking interaction. For the application, the magnetic PP-CMP was used for the enrichment of trace OH-PAHs in human urine of both smokers and nonsmokers in combination with high-performance liquid chromatography with fluorescence detection (HPLC-FLD). It showed good selectivity and excellent sensitivity to these OH-PAHs. Their detection limits were low and in the range of 0.01-0.08 μg·L(-1). The OH-PAHs were detected with different amounts from 0.054 to 0.802 μg·L(-1) in urine samples from smokers and nonsmokers. The recoveries were found to be 76.0%-107.8%. The results indicate that the magnetic PP-CMP offers an efficient enrichment method for trace OH-PAHs in human urine.

Combination of ESI and MALDI mass spectrometry for qualitative, semi-quantitative and in situ analysis of gangliosides in brain.

Gangliosides are a family of complex lipids that are abundant in the brain. There is no doubt the investigations about the distribution of gangliosides in brian and the relationship between gangliosides and Alzheimer’s disease is profound. However, these investigations are full of challenges due to the structural complexity of gangliosides. In this work, the method for efficient extraction and enrichment of gangliosides from brain was established. Moreover, the distribution of gangliosides in brain was obtained by matrix-assisted laser desorption ionization (MALDI) mass spectrometry imaging (MSI). It was found that 3-aminoquinoline (3-AQ) as matrix was well-suited for MALDI MS analysis of gangliosides in negative ion mode. In addition, the pretreatment by ethanol (EtOH) cleaning brain section and the addition of ammonium formate greatly improved the MS signal of gangliosides in the brain section when MALDI MSI analysis was employed. The distribution of ganliosides in cerebral cortex, hippocampus and cerebellum was respectively acquired by electrospray ionization (ESI) MS and MALDI MSI, and the data were compared for reliability evaluation of MALDI MSI. Further, applying MALDI MSI technology, the distribution of gangliosides in amyloid precursor protein transgenic mouse brain was obtained, which may provide a new insight for bioresearch of Alzheimer’s disease (AD).

Fast and efficient enrichment of functional ILC2 from human whole blood

Abstract Group 2 innate lymphoid cells (ILC2) are a functionally distinct subset of recently identified immune cells with important roles in type-2 immunopathologies such as allergies, asthma, helminth infections and other metabolic diseases. Studying these rare cells is challenging due to a lack of specific surface markers, and currently multicolor flow cytometric analysis and cell sorting are the only methods to characterize and isolate ILC2s. However, the scarcity of these cells makes flow cytometry time-consuming, expensive and often results in low purities and recoveries. Thus, better approaches for effective identification and isolation are essential to further understanding of ILC2 biology and function. We have developed a rapid and efficient method for enrichment of human ILC2 from whole blood. In brief, non-ILC2 cells in whole blood were crosslinked to red blood cells already present in the sample using RosetteSep™. The sample was then layered over Lymphoprep in a SepMate™ tube, spun at 1200 x g for 10 minutes (min), and the untouched, desired cells simply poured off. Cells were washed once and were then ready for subsequent analysis. Starting with only 0.01 – 0.07% in whole blood, ILC2 were enriched 350 ± 220 fold to 8.2 ± 6.8% in 35 min (means ± SD, n=17). Subsequent cell sorting from these pre-enriched samples was faster and yielded higher purity ILC2 than sorting from non-enriched controls (n=3, p<0.05 paired t test). Sorted ILC2, both pre-enriched and non-enriched controls, were cultured and stimulated, and secreted similar high levels of IL-13 as assessed by ELISA, indicating that these cells are functional. In summary, ILC2 pre-enrichment improves sorting efficiency, increases ILC2 purity, and maintains ILC2 functionality.

An efficient and rapid method for enrichment of lipophilic proteins from Mycobacterium tuberculosis H37Rv for two-dimensional gel electrophoresis.

Lipophilic proteome profiling is crucial because they have an anticipated role in biological processes and pathogenesis of Mycobacterium tuberculosis. These lipophilic proteins might be used as potential targets for the development of newer diagnostic markers and drug targets due to their association with membranes and drugs. We developed an efficient and rapid method to enrich the lipophilic proteins extraction from M. tuberculosis H37Rv for 2DE. In the extraction of lipophilic proteins, nonionic detergent (Triton X-100) was added in sonication buffer that augmented the solubilization of the proteins at the time of sonication. Enriched whole cell lysate was subjected to direct phase separation using Triton X-114, without the need for preisolation of membranes. In this study, we report that our optimized extraction buffer increased the lipophilic proteins extraction and their improved resolution on 2D gel up to two- to threefolds (quantitatively and qualitatively) as compared to standard extraction buffer. Some proteins were identified by MALDI-TOF/MS.

Sequential Elution from IMAC (SIMAC): An Efficient Method for Enrichment and Separation of Mono- and Multi-phosphorylated Peptides.

Phosphoproteomics relies on methods for efficient purification and sequencing of phosphopeptides from highly complex biological systems, especially when using low amounts of starting material. Current methods for phosphopeptide enrichment, e.g., Immobilized Metal ion Affinity Chromatography and titanium dioxide chromatography provide varying degrees of selectivity and specificity for phosphopeptide enrichment. The number of multi-phosphorylated peptides identified in most published studies is rather low. Here we describe a protocol for a strategy that separates mono-phosphorylated peptides from multiply phosphorylated peptides using Sequential elution from Immobilized Metal ion Affinity Chromatography. The method relies on the initial enrichment and separation of mono- and multi-phosphorylated peptides using Immobilized Metal ion Affinity Chromatography and a subsequent enrichment of the mono-phosphorylated peptides using titanium dioxide chromatography. The two separate phosphopeptide fractions are then subsequently analyzed by mass spectrometric methods optimized for mono-phosphorylated and multi-phosphorylated peptides, respectively, resulting in improved identification of especially multi-phosphorylated peptides from a minimum amount of starting material.

An efficient and target-oriented sample enrichment method for preparative separation of minor alkaloids by pH-zone-refining counter-current chromatography

An efficient and target-oriented sample enrichment method was established to increase the content of the minor alkaloids in crude extract by using the corresponding two-phase solvent system applied in pH-zone-refining counter-current chromatography. The enrichment and separation of seven minor indole alkaloids from Uncaria rhynchophylla (Miq.) Miq. ex Havil(UR) were selected as an example to show the advantage of this method. An optimized two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (3:7:1:9, v/v) was used in this study, where triethylamine (TEA) as the retainer and hydrochloric acid (HCl) as the eluter were added at the equimolar of 10mM. Crude alkaloids of UR dissolved in the corresponding upper phase (containing 10mM TEA) were extracted twice with lower phase (containing 10mM TEA) and lower phase (containing 10mM HCl), respectively, the second lower phase extract was subjected to pH-zone-refining CCC separation after alkalization and desalination. Finally, from 10g of crude alkaloids, 4g of refined alkaloids was obtained and the total content of seven target indole alkaloids was increased from 4.64% to 15.78%. Seven indole alkaloids, including 54mg isocorynoxeine, 21mg corynoxeine, 46mg isorhynchophylline, 35mg rhynchophylline, 65mg hirsutine, 51mg hirsuteine and 27mg geissoschizine methylether were all simultaneously separated from 2.5g of refined alkaloids, with the purity of 86.4%, 97.5%, 90.3%, 92.1%, 98.5%, 92.3%, and 92.8%, respectively. The total content and purities of the seven minor indole alkaloids were tested by HPLC and their chemical structures were elucidated by ESI-HRMS and 1H NMR.

Anchored multiplex PCR for targeted next-generation sequencing.

We describe a rapid target enrichment method for next-generation sequencing, termed anchored multiplex PCR (AMP), that is compatible with low nucleic acid input from formalin-fixed paraffin-embedded (FFPE) specimens. AMP is effective in detecting gene rearrangements (without prior knowledge of the fusion partners), single nucleotide variants, insertions, deletions and copy number changes. Validation of a gene rearrangement panel using 319 FFPE samples showed 100% sensitivity (95% confidence limit: 96.5-100%) and 100% specificity (95% confidence limit: 99.3-100%) compared with reference assays. On the basis of our experience with performing AMP on 986 clinical FFPE samples, we show its potential as both a robust clinical assay and a powerful discovery tool, which we used to identify new therapeutically important gene fusions: ARHGEF2-NTRK1 and CHTOP-NTRK1 in glioblastoma, MSN-ROS1, TRIM4-BRAF, VAMP2-NRG1, TPM3-NTRK1 and RUFY2-RET in lung cancer, FGFR2-CREB5 in cholangiocarcinoma and PPL-NTRK1 in thyroid carcinoma. AMP is a scalable and efficient next-generation sequencing target enrichment method for research and clinical applications.

Selective glycopeptide profiling by acetone enrichment and LC/MS

LC/MS is commonly used for site-specific glycosylation analysis of glycoproteins in cells and tissues. A limitation of this technique is the difficulty in acquiring reliable mass spectra for glycopeptides, mainly due to their high heterogeneity and poor hydrophobicity. Here, we establish a versatile method for efficient glycopeptide enrichment to acquire reliable mass spectra. Several lines of evidence using model glycoproteins suggest that our method is based on the different solubility between non-glycosylated and glycosylated peptides in acetone. We also provide data showing that the acetone-precipitated glycopeptide enrichment was successful in acquiring a more comprehensive MS/MS data set for the various glycoforms of each glycopeptide in crude human serum. We propose that this method is a powerful tool for the acquisition of reliable mass spectra from trace amounts of glycopeptides and an alternative to lectin affinity enrichment. In this study, we established a versatile method for glycopeptide enrichment to acquire reliable mass spectra because of the limitation of conventional enrichment methods, such as lectin affinity chromatography. Our enrichment method is capable of isolating glycopeptides from complex peptide mixtures such as crude serum.

Highly Efficient Enrichment Method for Glycopeptide Analyses: Using Specific and Nonspecific Nanoparticles Synergistically

We invented a new method for highly efficient and specific enrichment of glycopeptides using two different nanomaterials synergistically. One is boronic-acid-functionalized Fe3O4 nanoparticles, enriching glycopeptides through formation of cyclic boronate esters between the boronic acid groups and the cis-diol groups on glycopeptides. The other nanomaterial is conventional poly(methyl methacrylate) nanobeads, which have strong adsorption toward nonglycopeptides. By optimizing the proportion of these two materials, extremely high sensitivity and selectivity are achieved in analyzing the standard glycopeptides/nonglycopeptides mixture solutions. Since the washing step is not necessary for these conditions, the enrichment process is simplified and the recovery efficiency of target glycopeptides reaches 90%. Finally, this approach is successfully applied to analyze human serum with the sample volume as little as 1 μL, in which 147 different N-glycosylation peptides within 66 unique glycoproteins are identified. All these performances by the synergistic enrichment are much better than employing one specific enrichment agent alone.

Preparation of cysteine-click maltose modified silica as a hydrophilic interaction liquid chromatography material for the enrichment of glycopeptides

Because of the low abundance of glycoprotein and glycopeptide in complex biological samples, it is urgent to develop an efficient method for glycopeptide enrichment in comprehensive and in-depth glycoproteomes research. Herein, a novel hydrophilic silica was developed through surface modification with cysteine-click maltose (Cys-Mal@SiO2). The developed hydrophilic silica was packed into a solid phase extraction (SPE) column, and applied to the highly selective enrichment and identification of N-linked glycopeptides. The Cys-Mal@SiO2 demonstrated better identification capability over Cys@SiO2, Mal@SiO2 and commercial hydrophilic interaction liquid chromatography (HILIC) in glycopeptide enrichment due to the synergistic effect of the two kinds of hydrophilic molecules. In the selective enrichment of tryptic digest from human immunoglobulin G, glycopeptides with higher signal-to-noises were detected by Cys-Mal@SiO2. In addition, 1551 unique glycopeptides with 906 N-glycosylation sites from 466 different N-linked glycoproteins were identified from the proteins extracted from mouse liver after the enrichment with Cys-Mal@SiO2. In contrast, the numbers of identified glycopeptides, glycoproteins and N-glycosylation sites identified by Cys@SiO2 were 211, 67, 127 respectively less than by Cys-Mal@SiO2, and the corresponding numbers were 289, 76, 193 by Mal@SiO2. These results showed that the developed Cys-Mal@SiO2 is a promising affinity material for N-glycoproteomics research of real complex biological samples.

Preparation of titanium-grafted magnetic mesoporous silica for the enrichment of endogenous serum phosphopeptides

As one of the most important post-translational modifications, reversible phosphorylation of protein plays crucial roles in a large number of biological processes. Moreover, endogenous phosphopeptides are also associated with certain human diseases. An efficient enrichment and separation method is the premise for successful identification and quantification of phosphopeptides. In this work, titanium grafted magnetic mesoporous silica (Fe3O4@Ti-mSiO2) was developed and applied for the enrichment of endogenous phosphopeptides. Fe3O4@Ti-mSiO2 particles were prepared by grafting titanocene dichloride on the inner walls of magnetic mesoporous silica and then being calcined to remove cyclopentadienyl ligand. The physicochemical properties of the prepared materials were characterized by energy dispersive X-ray spectroscopy (EDX), nitrogen adsorption–desorption analysis, X-ray diffraction (XRD), transmission electron microscopy (TEM), scanning electron microscopy (SEM) and vibrating sample magnetometer (VSM). For selective enrichment of phosphopeptides, the prepared Fe3O4@Ti-mSiO2 particles were applied for tryptic digests of β-casein, mixtures of β-casein and bovine serum albumin (BSA), and low-fat milk. Finally, Fe3O4@Ti-mSiO2 was successfully applied for the enrichment of endogenous phosphopeptides from human serum.

Development of an immunomagnetic separation method for efficient enrichment of Escherichia coli O157:H7

Immunomagnetic separation uses antibody-coated paramagnetic particles to selectively bind and purify the target organisms from a comprehensive range of complex food matrices. Aim of this study was to develop and optimize an immunomagnetic separation method based on monoclonal antibody for efficient isolation of Escherichia coli O157:H7 in food samples. The key parameters for preparing the immunomagnetic beads; the coupling rate between monoclonal antibody and magnetic beads, additive amount of immunomagnetic beads, and magnetic separation times were optimized with different concentrations of E. coli O157:H7. Under optimized conditions, the capture efficiency (CE) was greater than 98% against 101–106 cells of E. coli O157:H7 with 0.05 mg immunomagnetic beads. The immunomagnetic beads exhibited high specific binding with E. coli O157:H7 strains (CE > 98%), and low binding with non-target bacteria (CE < 2%) except for S. aureus (CE = 23.6%). The capture efficiency of immunomagnetic beads against E. coli O157:H7 in ground beef and milk samples were 94.4% and 99.8%, respectively.

Microcavity Array System for Size-Based Enrichment of Circulating Tumor Cells from the Blood of Patients with Small-Cell Lung Cancer

In this study, we present a method for efficient enrichment of small-sized circulating tumor cells (CTCs) such as those found in the blood of small-cell lung cancer (SCLC) patients using a microcavity array (MCA) system. To enrich CTCs from whole blood, a microfabricated nickel filter with a rectangular MCA (10(4) cavities/filter) was integrated with a miniaturized device, allowing for the isolation of tumor cells based on differences in size and deformability between tumor and blood cells. The shape and porosity of the MCA were optimized to efficiently capture small tumor cells on the microcavities under low flow resistance conditions, while allowing other blood cells to effectively pass through. Under optimized conditions, approximately 80% of SCLC (NCI-H69 and NCI-H82) cells spiked in 1 mL of whole blood were successfully recovered. In clinical samples, CTCs were detectable in 16 of 16 SCLC patients. In addition, the number of leukocytes captured on the rectangular MCA was significantly lower than that on the circular MCA (p < 0.001), suggesting that the use of the rectangular MCA diminishes a considerable number of carryover leukocytes. Therefore, our system has potential as a tool for the detection of CTCs in small cell-type tumors and detailed molecular analyses of CTCs.

Abstract 428: Peptides Enrichment by Hydrophobic Interaction Chromatography for Detection of Oxidized Phospholipid-modified Protein Adducts

Oxidized phospholipids (oxPLs) accumulate at sites of oxidative stress and contribute significantly to atherosclerosis, acute inflammation, lung injury, and ischemia. Most oxPLs have electrophilic substituents and are highly likely to form covalent adducts with proteins, thus compromising protein function. Detection of covalent interaction between oxPLs and proteins could provide important information about the type of proteins preferentially modified by oxPLs and on the mechanism of interaction between proteins and oxPLs. These studies are very challenging due to low levels of oxPLs protein adducts. We have developed a highly efficient enrichment method for oxPLs-modified peptides that employs C18 solid phase extraction columns. This method takes advantage of the difference in polarity between oxPLs modified peptides and the corresponding unmodified peptides as well as other interfering compounds. Using this enrichment method, we have been able to detect very low levels of oxPLs modified peptides (&lt; 2 ppm) from tryptic digestion mixture of cell lysate using LC-MS. To demonstrate the performance of the method, we studied the interaction of apolipoprotein A-I (apoA-I) in high density lipoprotein with a specific oxPL (1-palmitoyl-2-(5’-oxo-valeroyl)-sn-glycero-3-phosphocholine, POVPC) at pathophysiological concentrations. We found that Lys 94, Lys 96 and Lys 195 are covalently modified by POVPC. Thus, the present study is the first to detected oxPL adducts of apoA-I in HDL.

Enrichment and antioxidant properties of flavone C-glycosides from trollflowers using macroporous resin

Orientin, vitexin and other flavone C-glycosides are functional ingredients abundant in trollflowers. In this study, an efficient method for enrichment of these ingredients from the flowers of Trollius chinensis Bunge was developed using macroporous resin. Separation characteristics of six typical macroporous resins were investigated by static adsorption/desorption and dynamic separation experiments, and HPD450 was selected as optimal one. Dynamic adsorption/desorption experiments on HPD450 columns were conducted to obtain the optimal parameters, followed by a scale-up experiment. Six fractions, TC-1–TC-3 together with their acid hydrolysates were further prepared to evaluate their antioxidant capacities by 2,2-diphenyl-1-picrylhydrazyl radical and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonate) radical cation scavenging assays. They all have notable concentration-dependent antioxidant activities, with TC-1 showing strong antioxidant capacity higher than orientin. The separation process was high-efficient, low-cost and environmental-friendly, which could afford a potential approach for industrial applications.