Multidimensional Liquid Chromatography Research Articles

Multiple, simultaneous, independent gradients for a versatile multidimensional liquid chromatography. Part II: Application 2: Computer controlled pH gradients in the presence of urea provide improved separation of proteins: Stability influenced anion and cation exchange chromatography

This paper details the use of a method of creating controlled pH gradients (pISep) to improve the separation of protein isoforms on ion exchange (IEX) stationary phases in the presence of various isocratic levels of urea. The pISep technology enables the development of computer controlled pH gradients on both cationic (CEX) and anionic (AEX) IEX stationary phases over the very wide pH range from 2 to 12. In pISep, titration curves generated by proportional mixing of the acidic and basic pISep working buffers alone, or in the presence of non-buffering solutes such as the neutral salt NaCl (0–1M), polar organics such as urea (0–8M) or acetonitrile (0–80 Vol%), can be fitted with high fidelity using high order polynomials which, in turn allows construction of a mathematical manifold %A (% acidic pISep buffer) vs. pH vs. [non-buffering solute], permitting precise computer control of pH and the non-buffering solute concentration allowing formation of dual uncoupled liquid chromatographic (LC) gradients of arbitrary shape (Hirsh and Tsonev, 2012 [1]). The separation of protein isoforms examined in this paper by use of such pH gradients in the presence of urea demonstrates the fractionation power of a true single step two dimensional liquid chromatography which we denote as Stability-Influenced Ion Exchange Chromatography (SIIEX). We present evidence that SIIEX is capable of increasing the resolution of protein isoforms difficult to separate by ordinary pH gradient IEX, and potentially simplifying the development of laboratory and production purification strategies involving on-column simultaneous pH and urea unfolding or refolding of targeted proteins. We model some of the physics implied by the dynamics of the observed protein fractionations as a function of both urea concentration and pH assuming that urea-induced native state unfolding competes with native state electrostatic interaction binding to an IEX stationary phase. Implications for in vivo protein-membrane interactions are discussed.

Characterization of the Molecular Mechanisms Underlying the Chronic Phase of Stroke in a Cynomolgus Monkey Model of Induced Cerebral Ischemia.

Stroke is one of the main causes of mortality and long-term disability worldwide. The pathophysiological mechanisms underlying this disease are not well understood, particularly in the chronic phase after the initial ischemic episode. In this study, a Macaca fascicularis stroke model consisting of two sample groups, as determined by MRI-quantified infarct volumes as a measure of the stroke severity 28 days after the ischemic episode, was evaluated using qualitative and quantitative proteomics analyses. By using multiple online multidimensional liquid chromatography platforms, 8790 nonredundant proteins were identified that condensed to 5223 protein groups at 1% global false discovery rate (FDR). After the application of a conservative criterion (5% local FDR), 4906 protein groups were identified from the analysis of cerebral cortex. Of the 2068 quantified proteins, differential proteomic analyses revealed that 31 and 23 were dysregulated in the elevated- and low-infarct-volume groups, respectively. Neurogenesis, synaptogenesis, and inflammation featured prominently as the cellular processes associated with these dysregulated proteins. Protein interaction network analysis revealed that the dysregulated proteins for inflammation and neurogenesis were highly connected, suggesting potential cross-talk between these processes in modulating the cytoskeletal structure and dynamics in the chronic phase poststroke. Elucidating the long-term consequences of brain tissue injuries from a cellular prospective, as well as the molecular mechanisms that are involved, would provide a basis for the development of new potentially neurorestorative therapies.

Phosphoproteome of crab-eating macaque cerebral cortex characterized through multidimensional reversed-phase liquid chromatography/mass spectrometry with tandem anion/cation exchange columns

A fully automated online multidimensional liquid chromatography (MDLC) platform featuring high-/low-pH reversed-phase (RP) dimensions and two other complementary—strong anion exchange (SAX) and strong cation exchange (SCX), respectively—chromatographic separations in tandem, with conventional offline titanium dioxide pre-enrichment, has been applied for the first global phosphopeptide identification from the macaque cerebral cortex in the presence of phosphatase inhibitors. Phosphorylation data interpretation, including site determination, and network construction have been performed: 14,338 distinct phosphopeptides in 7572 non-redundant phosphosites at 1% FDR were identified with 784 novel phosphorylation sites when mapping into the two most-curated public phosphorylation databases, PhosphoSitePlus (PSP) and Phospho.ELM (ELM), using probability-based placements. The net charges of both extremely acidic and basic phosphopeptides depend largely on the pH of the solvent, in turn impacting their retention and subsequent fractionation; the inclusion of the complementary SAX and SCX column chemistries after the high-pH RP dimension allowed effective retention and separation of net-negatively and −positively charged phosphopeptides, thereby leading to extended anionic and cationic phosphopeptide coverage from basophilic and acidophilic kinase substrates. A valuable protein interaction network of known and predicted motifs kinases was constructed from 3064 confident phosphorylation sites in the non-human primate’s brain.

Cell cycle-regulated ubiquitination of tankyrase 1 by RNF8 and ABRO1/BRCC36 controls the timing of sister telomere resolution.

Timely resolution of sister chromatid cohesion in G2/M is essential for genome integrity. Resolution at telomeres requires the poly(ADP-ribose) polymerase tankyrase 1, but the mechanism that times its action is unknown. Here, we show that tankyrase 1 activity at telomeres is controlled by a ubiquitination/deubiquitination cycle depending on opposing ubiquitin ligase and deubiquitinase activities. In late S/G2 phase, the DNA damage-responsive E3 ligase RNF8 conjugates K63-linked ubiquitin chains to tankyrase 1, while in G1 phase such ubiquitin chains are removed by BRISC, an ABRO1/BRCC36-containing deubiquitinase complex. We show that K63-linked ubiquitin chains accumulate on tankyrase 1 in late S/G2 to promote its stabilization, association with telomeres, and resolution of cohesion. Timing of this posttranslational modification coincides with the ATM-mediated DNA damage response that occurs on functional telomeres following replication in G2. Removal of ubiquitin chains is controlled by ABRO1/BRCC36 and occurs as cells exit mitosis and enter G1, ensuring that telomere cohesion is not resolved prematurely in S phase. Our studies suggest that a cell cycle-regulated posttranslational mechanism couples resolution of telomere cohesion with completion of telomere replication to ensure genome integrity.

Multidimensional Liquid Chromatography Coupled with Tandem Mass Spectrometry for Identification of Bioactive Fatty Acyl Derivatives.

Recognition of the contributions of lipids to cellular physiology, both as structural components of the membrane and as modulatory ligands for membrane proteins, has increased in recent years with the development of the biophysical and biochemical tools to examine these effects. Their modulatory roles in ion channels and transporters function have been extensively characterized, with the molecular mechanisms of these activities being the subject of intense scrutiny. The physiological significance of lipids in biochemistry is expanding as numerous fatty acyls are discovered to possess signaling properties. These bioactive lipids are often found in quantities of pmol/g of tissue and are co-extracted with numerous lipophilic molecules, making their detection and identification challenging. Common analytical methodologies involve chromatographic separation and mass spectrometric techniques; however, a single chromatographic step is typically ineffective due to the complexity of the biological samples. It is, therefore, essential to develop approaches that incorporate multiple dimensions of separation. Described in this manuscript are normal phase and reversed phase separation strategies for lipids that include detection of the bioactive primary fatty acid amides and N-acyl glycines via tandem mass spectrometry. Concerted utilization of these approaches are then used to separate and sensitively identify primary fatty acid amides extracted from homogenized tissue, using mouse brains as a test case.

Identification of three novel angiotensin-converting enzyme inhibitory peptides derived from cauliflower by-products by multidimensional liquid chromatography and bioinformatics

The aim of this paper was the development of an analytical strategy for the production of purified bioactive peptides from cauliflower waste proteins, by testing two different extraction protocols and screening different enzymes for protein hydrolysis. The purification of peptides was carried out by multidimensional liquid chromatography employing reversed phase chromatography and hydrophilic interaction chromatography; the resulting fractions were tested for antihypertensive and antioxidative activities. The most active ones were characterized by nano-liquid chromatography-tandem mass spectrometry and identified by database search. The identified peptides were further mined by in-silico analysis using PeptideRanker to ascribe a bioactivity rank to each peptide. Thus, six potential ACE-inhibitory peptides were synthesized and validated checking their retention times and fragmentation patterns for consistency. Pure peptide standards were finally in-vitro tested for the specific bioactivity. In this way, three novel ACE-inhibitory peptides were successfully identified and validated from cauliflower waste hydrolysate, showing good IC50 values.

Multiple, simultaneous, independent gradients for a versatile multidimensional liquid chromatography. Part II: Application 1 - Large increases in isoform resolution of human transferrin by use of dual simultaneous independent gradients of pH & acetonitrile on a mixed bed (anion exchange plus reversed phase) stationary phase

We have previously described a liquid chromatographic (LC) method for uncoupling controlled, wide range pH gradients and simultaneous controlled gradients of a non-buffering solute on ion exchange resins (Hirsh and Tsonev, 2012) [1]. Here we report the application of this two dimensional LC technique to the problem of resolving Human Transferrin (HT) isoforms. This important iron transporting protein should theoretically occur in several thousand glycoforms, but only about a dozen have been reported. Using dual simultaneous independent gradients (DSIGs) of acetonitrile (ACN) and pH on a mixed bed stationary phase (SP) consisting of a mixture of an anion exchange resin and a reversed phase (RP) resin we partially resolve about 60 isoforms. These are likely to be partially refolded glycoforms generated by interaction of HT with the highly hydrophobic RP SP, as well as distinct folded glycoforms. Thus this study should have interesting implications for both glycoform separation and the study of protein folding.

Abstract 5157: Quantitative proteomic analysis identifying Krt17 plays a critical role in areca nut inducing oral carcinogenesis

Abstract Areca nut is known a carcinogen for oral cancer in southeast Asia, however, the molecular mechanism leading to the malignancy is still unclear. To mimic the habit of areca nut chewing, our laboratory has established four oral cancer cell sublines (SAS, OECM1, K2, C9), chronically trained by areca nut extract (ANE). To elucidate the molecular basis of areca nut induced oral carcinogenesis, the differential proteomes between oral cancer cells and the ANE sublines were determined using the technique of isobaric mass tag (iTRAQ) labeling and multidimensional liquid chromatography- mass spectrometry (LC-MS/MS). Over thousand proteins were identified in four sublines, in which 194 proteins were found differential expressions in at least two ANE sublines. Bioinforatmic analysis revealed that these proteins participate in several pathways, with the regulation of epithelial to mesenchymal transition (EMT) most prominent. Fourteen proteins were confirmed differential expression in the ANE sublines, including Krt17. To shed more light on the mechanism of ANE induced carcinogenesis, Krt17 was further investigated. Knockdown Krt17 significantly suppressed ANE-induced cell growth, migration, and cell invasion, along with the modulation of EMT process. Furthermore, in a carcinogen-induced oral cancer mice model by 4NQO/arecoline, an active compound of ANE, Krt17 was found significantly up-regulated in all hyperplasia and carcinoma (p<0.001). In conclusion, we have identified proteome associated with chronic areca nut exposure in oral cancer cells. Krt17 was demonstrated contributing to areca nut induced oral malignancy. This study should attribute to risk assessment, disease prevention or other clinical applications of areca nut-induced oral cancer. Citation Format: Chang Hsu Chiang, Chih-Ching Wu, Ann-Joy Cheng. Quantitative proteomic analysis identifying Krt17 plays a critical role in areca nut inducing oral carcinogenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5157.

Purification and identification of endogenous antioxidant and ACE-inhibitory peptides from donkey milk by multidimensional liquid chromatography and nanoHPLC-high resolution mass spectrometry.

Donkey milk is a valuable product for the food industry due to its nutraceutical, nutritional, and functional properties. In this work, the endogenous peptides from donkey milk were investigated for their antioxidant and ACE-inhibitory activities, combining a two-dimensional peptide fractionation strategy with high-resolution mass spectrometry, bioinformatics analysis, and in vitro assays. After extraction, the endogenous peptides were fractionated twice, first by polymeric reversed phase and then by hydrophilic interaction chromatography. Fractions were screened for the investigated bioactivities and only the most active ones were finally analyzed by nanoRP-HPLC-MS/MS; this approach allowed to reduce the total number of possible bioactive sequences. Results were further mined by in silico analysis using PeptideRanker, BioPep, and PepBank, which provided a bioactivity score to the identified peptides and matched sequences to known bioactive peptides, in order to select candidates for chemical synthesis. Thus, five peptides were prepared and then compared to the natural occurring ones, checking their retention times and fragmentation patterns in donkey milk alone and in spiked donkey milk samples. Pure peptide standards were finally in vitro tested for the specific bioactivity. In this way, two novel endogenous antioxidant peptides, namely EWFTFLKEAGQGAKDMWR and GQGAKDMWR, and two ACE-inhibitory peptides, namely REWFTFLK and MPFLKSPIVPF, were successfully validated from donkey milk. Graphical Abstract Analytical workflow for purification and identification of bioactive peptides from donkey milk sample.

Proteome analysis for the global proteins in the jejunum tissues of enterotoxigenic Escherichia coli -infected piglets.

Enterotoxigenic Escherichia coli (ETEC) is a common cause of diarrhea in humans and livestock. In this study, isobaric tags for relative and absolute quantitation (iTRAQ) combined with multidimensional liquid chromatography (LC) and MS analysis was used for screening the differentially expressed proteins in piglet jejunum after ETEC infection. Totally 1,897 proteins were identified with quantitative information in piglet jejunum. We identified 92 differentially expressed proteins in ETEC-induced diarrhea, of which 30 were up regulated and 62 down regulated. Most of the differentially expressed proteins were involved in intestinal function of binding, metabolic process, catalytic activity and immune responses. The inhibition of intestinal immune responses in the jejunum in ETEC-induced diarrhea was also validated by immunobloting and RT-PCR. Our study is the first attempt to analyze the protein profile of ETEC-infected piglets by quantitative proteomics, and our findings could provide valuable information with respect to better understanding the host response to ETEC infection.

Proteome Profile and Quantitative Proteomic Analysis of Buffalo (Bubalusbubalis) Follicular Fluid during Follicle Development.

Follicular fluid (FF) accumulates in the antrum of the ovarian follicle and provides the microenvironment for oocyte development. FF plays an important role in follicle growth and oocyte maturation. The FF provides a unique window to investigate the processes occurring during buffalo follicular development. The observed low quality of buffalo oocytes may arise from the poor follicular microenvironment. Investigating proteins found in buffalo FF (BFF) should provide insight into follicular development processes and provide further understanding of intra-follicular maturation and oocytes quality. Here, a proteomic-based approach was used to analyze the proteome of BFF. SDS-PAGE separation combined with mass spectrometry was used to generate the proteomic dataset. In total, 363 proteins were identified and classified by Gene Ontology terms. The proteins were assigned to 153 pathways, including signaling pathways. To evaluate difference in proteins expressed between BFF with different follicle size (small, <4 mm; and large, >8 mm), a quantitative proteomic analysis based on multi-dimensional liquid chromatography pre-fractionation tandem Orbitrap mass spectrometry identification was performed. Eleven differentially expressed proteins (six downregulated and five upregulated in large BFF) were identified and assigned to a variety of functional processes, including serine protease inhibition, oxidation protection and the complement cascade system. Three differentially expressed proteins, Vimentin, Peroxiredoxin-1 and SERPIND1, were verified by Western blotting, consistent with the quantitative proteomics results. Our datasets offers new information about proteins present in BFF and should facilitate the development of new biomarkers. These differentially expressed proteins illuminate the size-dependent protein changes in follicle microenvironment.

Comparative proteomic analysis of CD34+ cells in bone marrow between severe aplastic anemia and normal control

Severe aplastic anemia (SAA) is an autoimmune disease with destruction of hematopoietic cells by activated T lymphocytes. However, the precise mechanism of cytotoxicity T cells recognizing and attacking CD34+ cells remains unclear. Here, we investigated the proteome of CD34+ cells in SAA patients to further explore the pathogenesis of SAA. CD34+ cells from 29 SAA patients and 20 health controls were isolated by magnetic activated cell sorting. The protein of CD34+ cells were examined by iTRAQ labeling combination of multidimensional liquid chromatography and tandem mass spectrometry. A total of 156 differential expression proteins in CD34+ cells were identified. Compared with health controls, 53 proteins were up-regulated and 103 proteins were down-regulated in SAA patients. Specifically, abnormal expression of proteasome subunits, histone variants, dolichyl-diphosphooligosaccharide–protein glycosyltransferase subunit (DAD1) and ATPase inhibitor, mitochondrial isoform 1 precursor(IF1) may relate to the hyperfunction of immune responses and excessive apoptosis of SAA CD34+ cells.

Group-type characterization of crude oil and bitumen. Part I: Enhanced separation and quantification of saturates, aromatics, resins and asphaltenes (SARA)

Compositional analysis of crude oils is very important along the entire value chain of the oil industry, from upstream to midstream and downstream operations. An essential step of this analysis is to separate crude oil into fractions of saturate hydrocarbon (saturates), aromatic hydrocarbon (aromatics), resins, and asphaltenes (SARA). Though a number of methods have been developed for SARA separation, traditional methods involve significant drawbacks that include, but are not limited to, inconsistent results among labs and methods, extensive cross-contamination, and poor analytical precision. Here, we report on a novel automated multi-dimensional high performance liquid chromatography (AMD-HPLC) approach to enhance group-type characterization of crude oil or bitumen. The AMD-HPLC system is fully automated and developed based on a combination of adsorption and partition chromatography. It is equipped with a cyano column and a silica column. Each set of SARA columns is connected in series via multi-position valves that allow a given column to be inserted “in” or to be bypassed “out” of the flow path of the mobile phase at pre-set times in the process. In practice, a bitumen or oil sample of 0.02g is dissolved in a solvent mixture of 15% methanol, 15% acetone, and 70% chloroform, and injected into the instrument. The sample is fractionated into the four groups by selective retention through interactions with the solvent mobile phases and the column substrates. The instrument then resets and is ready to start the next run. The entire analysis cycle requires about 60min per run, including system re-conditioning. Based on its wide application at our laboratory, the AMD-HPLC is characterized by high efficiency, elimination of cross-contamination, and high reproducibility – when compared to traditional methods.

Exploration of Molecular Network Variations in Different Subtypes of Human Non-functional Pituitary Adenomas.

Exploration of Molecular Network Variations in Different Subtypes of Human Non-functional Pituitary Adenomas.

Application of Comprehensive Two-Dimensional Liquid Chromatography for Carotenoid Analysis in Red Mamey (Pouteria sapote) Fruit

One-dimensional liquid chromatography is widely applied to the analytical separation of various phytochemicals, including carotenoids; however, sometimes it does not provide sufficient resolution power for the analyte separation in very complex real-world samples. Multidimensional chromatography is a technique that may provide the required analytical separation power to tackle such a task. The aim of this work was to apply comprehensive multidimensional two-dimensional liquid chromatography, coupled to photodiode array and atmospheric pressure chemical ionization mass spectrometry detection, to the free carotenoid separation in red mamey (Pouteria sapote) fruit for the first time, using ultra high pressure liquid chromatography conditions in the second dimension separation. A comparison between one- and two-dimensional separation of the same matrix is shown, and a total of 23 different carotenoids belonging to 17 different carotenoid chemical classes were separated in the double orthogonal separation and identification system; moreover, a hypothesis for the possible detection of a new carotenoid is also reported for the first time.

Group-type characterization of crude oil and bitumen. Part II: Efficient separation and quantification of normal-paraffins iso-paraffins and naphthenes (PIN)

The SARA (saturates, aromatics, resins, and asphaltenes) separation and quantification have been widely used for characterizing the composition of crude oil and bitumen. The saturate hydrocarbons are the most valuable fraction of the oil industry and contain vital geochemical information for determining the origin and formation of petroleum fluids and for assessing the hydrocarbon potential and sedimentary environment of source rocks. It is therefore necessary to further separate the saturate fraction into normal paraffins, iso-paraffins and naphthenes (PIN fractions). Nevertheless, the convenient approach of PIN separation has never been reported on in the literature. Here, we present an efficient method that allows us to further separate the saturate fraction of crude oil or bitumen into PIN fractions through the automated multi-dimensional high performance liquid chromatography system (AMD-HPLC). In essence, the system is equipped with a “PIN1” column (Silicon: S115) packed with a 5.4Å molecular sieve and a “PIN2” column (Silicon: 50%S115+50%S130) packed with a 6.2Å molecular sieve. Each set of columns is connected in a series via multi-position valves that allow a given column to be inserted “in” or to be by-passed “out” of the flow path of the mobile phase at pre-set times in the process. When use iso-octane and n-octane or their mixture as mobile phases, the system separates the saturate hydrocarbon into PIN fractions simply through three steps under a temperature range of 25–140°C. An entire PIN analysis cycle takes nearly 60min, including system re-conditioning. The separated PIN fractions can be quantified using either gravimetric method or evaporative light scattering detector (ELSD) technique. Experimental results exhibit excellent qualitative and quantitative capability.

Proteomic Analysis of Protein Turnover by Metabolic Whole Rodent Pulse-Chase Isotopic Labeling and Shotgun Mass Spectrometry Analysis.

The analysis of protein half-life and degradation dynamics has proven critically important to our understanding of a broad and diverse set of biological conditions ranging from cancer to neurodegeneration. Historically these protein turnover measures have been performed in cells by monitoring protein levels after "pulse" labeling of newly synthesized proteins and subsequent chase periods. Comparing the level of labeled protein remaining as a function of time to the initial level reveals the protein's half-life. In this method we provide a detailed description of the workflow required for the determination of protein turnover rates on a whole proteome scale in vivo. Our approach starts with the metabolic labeling of whole rodents by restricting all the nitrogen in their diet to exclusively nitrogen-15 in the form of spirulina algae. After near complete organismal labeling with nitrogen-15, the rodents are then switched to a normal nitrogen-14 rich diet for time periods of days to years. Tissues are harvested, the extracts are fractionated, and the proteins are digested to peptides. Peptides are separated by multidimensional liquid chromatography and analyzed by high resolution orbitrap mass spectrometry (MS). The nitrogen-15 containing proteins are then identified and measured by the bioinformatic proteome analysis tools Sequest, DTASelect2, and Census. In this way, our metabolic pulse-chase approach reveals in vivo protein decay rates proteome-wide.

Application of three-dimensional liquid chromatography for quantification of 2-acetyl-4-(1,2,3,4-tetrahydroxybutyl)imidazole (THI) in caramel colours

This study described a new method to quantify THI in caramels colours without sample preparation thanks to the separation power of multidimensional liquid chromatography.

Functional Proteomics of Regulatory and Epigenetic Signaling in Normal and Malignant Hematopoiesis

Post-translational modifications of proteins are increasingly recognized as key regulators of functional complexes assembly and cellular enzymes activity, processes that control both normal and aberrant cell growth and development. To enable comprehensive, sensitive and quantitative analysis of these processes, we adapted recently developed high-efficiency nanoscale multidimensional liquid chromatography with high-resolution Orbitrap mass spectrometry. Termed accumulated ion monitoring (AIM), this mass spectrometry method achieved nearly 7 orders of magnitude of quantitative accuracy and absolute limit of detection of 600 molecules per scan, enabling the study of rare cell populations (Fig. 1). We leveraged AIM mass spectrometry to develop a panel of 1583 synthetic reference peptides, based on global and published proteomics maps of normal human and leukemia cells. This Quantitative Cell Proteomics Atlas (QCPA) profiles 384 key effectors of cell surface signaling, proliferation, quiescence, stress response, and epigenetic control of gene expression (Fig. 2). QCPA enables unprecedented accuracy and sensitivity for the functional analysis of rare cells, with focus on protein regulation via phosphorylation, acetylation and methylation, as well as on concentration (http://alexkentsis.net/qcpa). By using functional proteomics profiling of primary human CD34+ and acute myeloid leukemia (AML) cells, we identify new pathways controlling erythrocyte differentiation and AML therapy resistance, as facilitated by a newly developed program ProteoModlR (http://github.com/kentsisresearchgroup/ProteoModlR). AIM mass spectrometry and QCPA functional proteomics are rapidly adaptable and generalizable tools for the investigation of regulatory and epigenetic signaling in normal and diseased cells. [Display omitted] [Display omitted] DisclosuresBauer:Editas Medicine: Consultancy; Biogen: Research Funding.

Multidimensional preparative liquid chromatography to isolate flavonoids from bergamot juice and evaluation of their anti-inflammatory potential.

Important objectives of a high-performance liquid chromatography preparative process are: purity of products isolated, yield, and throughput. The multidimensional preparative liquid chromatography method used in this work was developed mainly to increase the throughput; moreover purity and yield are increased thanks to the automated collection of the molecules based on the intensity of a signal generated from the mass spectrometer detector, in this way only a specific product can be targeted. This preparative system allowed, in few analyses both in the first and second dimensions, the isolation of eight pure compounds present at very different concentration in the original sample with high purity (>95%) and yield, which showed how the system is efficient and versatile. Pure molecules were used to validate the analytical method and to test the anti-inflammatory and antiproliferative potential of flavonoids. The contemporary presence, in bergamot juice, of all the flavonoids together increases the anti-inflammatory effect with respect to the single compound alone.