Tailored Polymer-Based Selective Extraction of Lipid Mediators from Biological Samples.

Lipid mediators, small molecules involved in regulating inflammation and its resolution, are a class of lipids of wide interest as their levels in blood and tissues may be used to monitor health and disease states or the effect of new treatments. These molecules are present at low levels in biological samples, and an enrichment step is often needed for their detection. We describe a rapid and selective method that uses new low-cost molecularly imprinted (MIP) and non-imprinted (NIP) polymeric sorbents for the extraction of lipid mediators from plasma and tissue samples. The extraction process was carried out in solid-phase extraction (SPE) cartridges, manually packed with the sorbents. After extraction, lipid mediators were quantified by liquid chromatography-tandem mass spectrometry (LC-MSMS). Various parameters affecting the extraction efficiency were evaluated to achieve optimal recovery and to reduce non-specific interactions. Preliminary tests showed that MIPs, designed using the prostaglandin biosynthetic precursor arachidonic acid, could effectively enrich prostaglandins and structurally related molecules. However, for other lipid mediators, MIP and NIP displayed comparable recoveries. Under optimized conditions, the recoveries of synthetic standards ranged from 62% to 100%. This new extraction method was applied to the determination of the lipid mediators concentration in human plasma and mouse tissues and compared to other methods based on commercially available cartridges. In general, the methods showed comparable performances. In terms of structural specificity, our newly synthesized materials accomplished better retention of prostaglandins (PGs), hydroxydocosahexaenoic acid (HDoHE), HEPE, hydroxyeicosatetraenoic acids (HETE), hydroxyeicosatrienoic acid (HETrE), and polyunsaturated fatty acid (PUFA) compounds, while the commercially available Strata-X showed a higher recovery for dihydroxyeicosatetraenoic acid (diHETrEs). In summary, our results suggest that this new material can be successfully implemented for the extraction of lipid mediators from biological samples.

Open tubular lab-on-column/mass spectrometry for targeted proteomics of nanogram sample amounts.

A novel open tubular nanoproteomic platform featuring accelerated on-line protein digestion and high-resolution nano liquid chromatography mass spectrometry (LC-MS) has been developed. The platform features very narrow open tubular columns, and is hence particularly suited for limited sample amounts. For enzymatic digestion of proteins, samples are passed through a 20 µm inner diameter (ID) trypsin + endoproteinase Lys-C immobilized open tubular enzyme reactor (OTER). Resulting peptides are subsequently trapped on a monolithic pre-column and transferred on-line to a 10 µm ID porous layer open tubular (PLOT) liquid chromatography LC separation column. Wnt/ß-catenein signaling pathway (Wnt-pathway) proteins of potentially diagnostic value were digested+detected in targeted-MS/MS mode in small cell samples and tumor tissues within 120 minutes. For example, a potential biomarker Axin1 was identifiable in just 10 ng of sample (protein extract of ∼1,000 HCT15 colon cancer cells). In comprehensive mode, the current OTER-PLOT set-up could be used to identify approximately 1500 proteins in HCT15 cells using a relatively short digestion+detection cycle (240 minutes), outperforming previously reported on-line digestion/separation systems. The platform is fully automated utilizing common commercial instrumentation and parts, while the reactor and columns are simple to produce and have low carry-over. These initial results point to automated solutions for fast and very sensitive MS based proteomics, especially for samples of limited size.

Integrated enzyme reactor and high resolving chromatography in "sub-chip" dimensions for sensitive protein mass spectrometry.

Reliable, sensitive and automatable analytical methodology is of great value in e.g. cancer diagnostics. In this context, an on-line system for enzymatic cleavage of proteins, subsequent peptide separation by liquid chromatography (LC) with mass spectrometric detection has been developed using "sub-chip" columns (10-20 µm inner diameter, ID). The system could detect attomole amounts of isolated cancer biomarker progastrin-releasing peptide (ProGRP), in a more automatable fashion compared to previous methods. The workflow combines protein digestion using an 20 µm ID immobilized trypsin reactor with a polymeric layer of 2-hydroxyethyl methacrylate-vinyl azlactone (HEMA-VDM), desalting on a polystyrene-divinylbenzene (PS-DVB) monolithic trap column, and subsequent separation of resulting peptides on a 10 µm ID (PS-DVB) porous layer open tubular (PLOT) column. The high resolution of the PLOT columns was maintained in the on-line system, resulting in narrow chromatographic peaks of 3-5 seconds. The trypsin reactors provided repeatable performance and were compatible with long-term storage.

Large volume injection of aqueous peptide samples on a monolithic silica based zwitterionic-hydrophilic interaction liquid chromatography system for characterization of posttranslational modifications.

Zwitterionic-hydrophilic interaction liquid chromatography (HILIC) has been found very appropriate for separation of polar compounds and peptides with post-translational modifications (PTMs) such as phosphorylation and glycosylation. In this study, a column switching system based on zwitterionic-HILIC silica based monolith columns was used for enrichment and separation of peptides and characterization of N-linked glycosylation by higher-energy collisional dissociation (HCD) Orbitrap mass spectrometry (MS). Peptides were found to be retained on a zwitterionic-HILIC precolumn, even in an aqueous buffer due to electrostatic interactions. Thus, a novel approach of using a zwitterionic-HILIC precolumn, for introduction of an aqueous sample such as a tryptic digest, followed by HILIC separation of the peptides is presented. The repeatability and loadability of the zwitterionic-HILIC-zwitterionic-HILIC column switching system were explored using a tryptic digest of transferrin and a mixture of six proteins. The column switching system was furthermore used to enrich and separate a tryptic digested rat liver extract gel fraction, where in total 48 peptides corresponding to 14 proteins were identified. N-linked glycoforms were also identified, both in the standard test proteins (transferrin and six protein mixture digest) and the rat liver extract fraction. In all cases, the identified N-linked glycoforms were identified at the end of the gradient, at high aqueous buffer content in the mobile phase, showing the suitability of the developed method for characterization of glycosylated peptides in aqueous samples.

Separation optimization of long porous-layer open-tubular columns for nano-LC-MS of limited proteomic samples.

The single-run resolving power of current 10 µm id porous-layer open-tubular (PLOT) columns has been optimized. The columns studied had a poly(styrene-co-divinylbenzene) porous layer (~0.75 µm thickness). In contrast to many previous studies that have employed complex plumbing or compromising set-ups, SPE-PLOT-LC-MS was assembled without the use of additional hardware/noncommercial parts, additional valves or sample splitting. A comprehensive study of various flow rates, gradient times, and column length combinations was undertaken. Maximum resolution for <400 bar was achieved using a 40 nL/min flow rate, a 400 min gradient and an 8 m long column. We obtained a 2.3-fold increase in peak capacity compared to previous PLOT studies (950 versus previously obtained 400, when using peak width = 2σ definition). Our system also meets or surpasses peak capacities obtained in recent reports using nano-ultra-performance LC conditions or long silica monolith nanocolumns. Nearly 500 proteins (1958 peptides) could be identified in just one single injection of an extract corresponding to 1000 BxPC3 beta catenin (-/-) cells, and ~1200 and 2500 proteins in extracts of 10,000 and 100,000 cells, respectively, allowing detection of central members and regulators of the Wnt signaling pathway.

High sensitivity measurements of active oxysterols with automated filtration/filter backflush-solid phase extraction-liquid chromatography-mass spectrometry.

Oxysterols are important in numerous biological processes, including cell signaling. Here we present an automated filtration/filter backflush-solid phase extraction-liquid chromatography-tandem mass spectrometry (AFFL-SPE-LC-MS/MS) method for determining 24-hydroxysterol and the isomers 25-hydroxycholesterol and 22S-hydroxycholesterol that enables simplified sample preparation, high sensitivity (~25 pg/mL cell lysis sample) and low sample variability. Only one sample transfer step was required for the entire process of cell lysis, derivatization and determination of selected oxysterols. During the procedure, autoxidation of cholesterol, a potential/common problem using standard analytical methods, was found to be negligible. The reversed phase AFFL-SPE-LC-MS/MS method utilizing a 1mm inner diameter column was validated, and used to determine levels of the oxysterol analytes in mouse fibroblast cell lines SSh-LII and NIH-3T3, and human cancer cell lines, BxPC3, HCT-15 and HCT-116. In BxPC3 cells, the AFFL-SPE-LC-MS/MS method was used to detect significant differences in 24S-OHC levels between vimentin+ and vimentin- heterogenous sub-populations. The methodology also allowed monitoring of significant alterations in 24S-OHC levels upon delivery of the Hedgehog (Hh) antagonist MS-0022 in HCT-116 colorectal carcinoma cell lines.

High efficiency, high temperature separations on silica based monolithic columns.

The effect of temperature on separation using reversed-phase monolithic columns has been investigated using a nano-LC pumping system for gradient separation of tryptic peptides with MS detection. A goal of this study was to find optimal conditions for high-speed separations. The chromatographic performance of the columns was evaluated by peak capacity and peak capacity per time unit. Column lengths ranging from 20 to 100 cm and intermediate gradient times from 10 to 30 min were investigated to assess the potential of these columns in a final step separation, e.g. after fractionation or specific sample preparation. Flow rates from 250 to 2000 nL/min and temperatures from 20 to 120°C were investigated. Temperature had a significant effect on fast separations, and a flow rate of 2000 nL/min and a temperature of 80°C gave the highest peak capacity per time unit. These settings produced 70% more protein identifications in a biological sample compared to a conventional packed column. Alternatively, an equal amount of protein identifications was obtained with a 40% reduction in run time compared to the conventional packed column.

Critical assessment of accelerating trypsination methods.

In LC-MS based proteomics, several accelerating trypsination methods have been introduced in order to speed up the protein digestion, which is often considered a bottleneck. Traditionally and most commonly, due to sample heterogeneity, overnight digestion at 37 °C is performed in order to digest both easily and more resistant proteins. High efficiency protein identification is important in proteomics, hours with LC-MS/MS analysis is needless if the majority of the proteins are not digested. Based on preliminary experiments utilizing some of the suggested accelerating methods, the question of whether accelerating digestion methods really provide the same protein identification efficiency as the overnight digestion was asked. In the present study we have evaluated four different accelerating trypsination methods (infrared (IR) and microwave assisted, solvent aided and immobilized trypsination). The methods were compared with conventional digestion at 37 °C in the same time range using a four protein mixture. Sequence coverage and peak area of intact proteins were used for the comparison. The accelerating methods were able to digest the proteins, but none of the methods appeared to be more efficient than the conventional digestion method at 37 °C. The conventional method at 37 °C is easy to perform using commercially available instrumentation and appears to be the digestion method to use. The digestion time in targeted proteomics can be optimized for each protein, while in comprehensive proteomics the digestion time should be extended due to sample heterogeneity and influence of other proteins present. Recommendations regarding optimizing and evaluating the tryptic digestion for both targeted and comprehensive proteomics are given, and a digestion method suitable as the first method for newcomers in comprehensive proteomics is suggested.

Separation of intact proteins on porous layer open tubular (PLOT) columns.

Porous layer open tubular (PLOT) polystyrene divinylbenzene columns have been used for separating intact proteins with gradient elution. The 10 microm I.D. x 3 m columns were easily coupled to standard liquid chromatography-mass spectrometry (LC-MS) instrumentation with commercially available fittings. Standard proteins separated on PLOT columns appeared as narrow and symmetrical peaks with good resolution. Average peak width increased linearly with gradient time (tG) from 0.14 to 0.33 min (tG 20 and 120 min, respectively) using a 3 m column. With shorter columns, peak widths were larger and increased more steeply with gradient time. Theoretical peak capacity (nc) increased with column length (tested up to 3 m). The nc increased with tG until a plateau was reached. The highest peak capacity achieved (nc=185) was obtained with a 3 m column, where a plateau was reached with tG 90 min. The within- and between column retention time repeatabilities were below 0.6% and below 2.5% (relative standard deviation, RSD), respectively. The carry-over following injection of 0.5 ng per protein was less than 1.1%. The retention time dependence on column temperature was investigated in the range 20-50 degrees C. Proteins in a skimmed milk sample were separated using the method.

Determination of thromboxanes, leukotrienes and lipoxins using high-temperature capillary liquid chromatography-tandem mass spectrometry and on-line sample preparation.

An on-line strong cation-exchange (SCX)-reversed-phase (RP) capillary liquid chromatographic (cLC) method with ion-trap tandem mass spectrometric (IT-MS/MS) detection for the simultaneous determination of thromboxane (TX) B(2), TXB(3), leukotriene (LT) B(4), LTD(4) and lipoxin (LX) A(4) in cell culture supernatants was developed and validated. In the present method, a high temperature (70 degrees C) was used for the separation on the analytical column to obtain efficient chromatography of the thromboxanes. An on-line sample preparation was performed, where peptides/proteins contained in the matrix were removed by the SCX column. Sample pre-treatment included dilution and filtration, and the analysis time including all sample preparation steps was 60min per sample. Limits of detection in the range of 1-4ng/mL cell culture supernatant, recoveries between 30% and 100%, within day precisions of less than 20% RSD and between day precisions of less than 30% RSD were obtained. Human mesenchymal stem cells (hMSCs) were stimulated with cytokine-containing supernatants derived from activated human T lymphocytes, and thromboxane, leukotriene and lipoxin production was analysed using the developed method. TXB(2) was found in cultures from both non-differentiated and differentiated hMSCs that were stimulated with a cytokine-containing supernatant obtained from activated T-cells.

On-Line multitasking analytical proteomics: how to separate, reduce, alkylate and digest whole proteins in an on-Line multidimensional chromatography system coupled to MS.

An on-Line multidimensional system has been developed, consisting of pH gradient strong anion exchange chromatography of native proteins in the first dimension with subsequent trapping and on-column reduction/alkylation on C4 trap columns and RP separation of the alkylated proteins in the second dimension followed by on-column tryptic digestion and electrospray MS detection. The system was evaluated using model proteins and a human urine sample. Compared to the commonly used in-solution alkylation method, the developed on-column method provides an equivalent efficiency. The recovery from the C4 trap columns of the alkylated proteins relative to the native state was from 94 to 102%. On-column tryptic digestion was satisfactory for many, but not for all proteins. The whole analytical procedure was performed on-Line with packed capillary columns for a total time of 320 min for the first ion exchange fraction, with additional 60 min for each subsequent fraction.

Detecting pM concentrations of prostaglandins in cell culture supernatants by capillary SCX-LC-MS/MS.

A highly sensitive, improved online strong cation exchange (SCX)--RP capillary liquid chromatographic (cLC) method with IT mass spectrometric (IT-MS/MS) detection for the simultaneous determination of prostaglandin (PG)A(1), PGD(2), PGE(1), PGE(2), PGF(2alpha), 8-iso-(8i)PGF(2alpha), 6-keto-(6k)PGF(1alpha), and 15-Delta(12, 14)-deoxy-PGJ(2) (15dPGJ(2)) in cell culture supernatants was developed and validated. Pretreatment of the cell culture supernatants included only dilution and filtration, and the analysis time including all sample preparation steps was 60 min per sample. Peptides/proteins contained in the matrix were removed by the SCX column. LODs in the range of 8-44 pg/mL (25-120 pM) cell culture supernatant were obtained. Excellent linearity (R(2) > 0.99) and satisfactory recoveries and within- and between-day precisions were obtained. Human mesenchymal stem cells (hMSCs) were stimulated with tumor necrosis factor alpha (TNFalpha) or TNFalpha/IL-17, and PG production was analyzed using the developed method. The four PGs, 6kPGF(1a), PGF(1a), PGE(2), and PGE(1 )were detected both in nonstimulated and stimulated cells. The amount of PG produced by the cell increased when the cell was stimulated.

Sensitive determination of a glyoxal-DNA adduct biomarker candidate by column switching capillary liquid chromatography electrospray ionization mass spectrometry.

A method based on column switching packed capillary liquid chromatography electrospray mass spectrometry has been developed for the determination of the adduct glyoxal-deoxyguanosine, a biomarker candidate for the assessment of glyoxal exposure, in DNA hydrolysate solutions. Microgram amounts of DNA were isolated and enzymatically hydrolyzed to deoxyribonucleosides, prior to ultrafiltration and subsequent dilution to a sample solution consisting of water-acetonitrile-formic acid (98 : 2 : 0.2, v/v). The sample solution was loaded onto a 1 mm I.D. x 5 mm Hypercarb (5 mum) porous graphitic carbon trap column for analyte enrichment using an injection volume of 200 mul, and was subsequently back-flushed onto a 0.30 mm I.D. x 150 mm Lichrospher diol (5 mum) analytical column. The samples were loaded with a flow rate of 40 mul min(-1) and glyoxal-deoxyguanosine was desorbed from the trap column and eluted with an isocratic mobile phase consisting of water-acetonitrile-formic acid (50 : 50 : 0.2, v/v) at a flow rate of 5 mul min(-1). Mass spectrometric determination of glyoxal-deoxyguanosine was obtained by multiple reaction monitoring of the transition [M + H](+)m/z 326 --> m/z 210. The method was evaluated over the concentration range 0.25-50 ng ml(-1) of glyoxal-deoxyguanosine in the hydrolysate of 5 mug DNA. The method was linear with a correlation coefficient of 0.9998 in this range. The within-day (n = 6) and between-day (n = 6) precisions were determined as 1.2-11% and 1.4-11% RSD, respectively, and the recovery was close to 100%. The mass limit of detection was 15 pg, corresponding to a concentration limit of detection of 75 fg mul(-1) DNA hydrolysate solution, corresponding to 48 adducts per 10(6) normal nucleosides. The method was applied for the determination of glyoxal-deoxyguanosine in DNA hydrolysate solutions of calf thymus DNA and cell cultures after reaction or incubation with glyoxal.

Improving the resolution of neuropeptides in rat brain with on-line HILIC-RP compared to on-line SCX-RP.

Our two already established on-line 2-D LC systems, a strong cation exchange-RP chromatography (SCX-RP) system and a hydrophilic interaction LC (HILIC)-RP 2-D LC system, were compared to explore which system is best suited for our further studies of differences in cerebral neuropeptide expression as a function of hypoxia-caused stress. The same mass spectrometer and database search parameters were applied in both systems. In total, 19 first dimension fractions were collected with the novel on-line HILIC-RP system, including a Hypercarb SPE column that was applied to trap the compounds not retained on a Kromasil C18 enrichment column. In contrast, six fractions were collected in the SCX-RP method, due to practical limitations of this traditional on-line 2-D LC system. With the on-line HILIC-RP system three times more peaks were detected. It was observed that most of the compounds eluted in the first two fractions in the SCX-RP method, while in the 2-D HILIC-RP method there seemed to be no correlation between peaks detected and fraction number. Thus, from this systematic study it seems that on-line HILIC-RP chromatography is the method of choice for comparative peptidomics of cerebral neuropeptides in future studies.