TFA In Water Research Articles

Application of HPLC-ELSD for the Quantification of 5-Aminolevulinic Acid after Penetration into Human Skin Ex Vivo

5-Aminolevulinic acid (5-ALA) has been used for treatment of different skin diseases (e.g., skin cancer, actinic keratosis (AK), psoriasis, and acne). The quality of the treatment is directly associated to the amount of 5-ALA that penetrates into skin. 5-ALA was extracted from skin samples after performing the experiment with Bronaugh cells for 4 hours. Two methods were developed by applying different column systems and mobile phase compositions: YMC-Pack Hydrosphere C18 column (250 × 4 mm, 5 µm) in series with Hichrom Hypersil H5ODS (150 × 4 mm, 5 µm), mobile phase consisted of 0.1% TFA in water with 2% of ACN (method A); ACE 3AQ column (150 × 4 mm, 3 µm) eluted with 0.05% TFA in water (method B). Exploratory fluorimetric analysis requiring pre-column or post-column derivatization did not warrant the expectations of simple and effective analysis; therefore, ELS detection for 5-ALA was considered. Parallel chromatography was applied for double-validation of the methods in order to correctly evaluate the possibility of ELSD application for 5-ALA detection and the quantification amount in skin extracts. The retention time and adequate lowest limit of quantification (LLOQ) of 5-ALA were determined in both methods: 5.5 min and 20 µg/ml (method A); 6.2 min and 8.4 µg/ml (method B). Both HPLC-ELSD methods proved to be simple, accurate, precise, reliable, and showed reproducible values in the concentration range of 20–200 µg/ml in human skin extracts.

Purification, antioxidant activity and protein-precipitating capacity of punicalin from pomegranate husk

Punicalin is a kind of ellagitannin, existing in pomegranate husk, and has shown remarkable biological activities. A rapid and large-scale separation method of punicalin from pomegranate husk was established, using medium pressure liquid chromatography (MPLC). The optimal mobile phase consisted of 5% methanol and 0.1% (v/v) TFA in water, and the optimal loading amount and flow rate were 1.0g and 80ml/min, respectively. Under this condition, 339mg of 95.9% punicalin could be obtained in 40min. 59.7mg of 78.0% gallic acid could be separated simultaneously. This method was practical for industrial utilisation of pomegranate husk. Afterwards, the antioxidant and protein-precipitating capacities of the purified punicalin, together with punicalagin, were evaluated. Results showed that punicalin had strong antioxidant activity, and it exhibited a low affinity for protein. This suggested that the antioxidant of punicalin would not be greatly masked by tannin–protein precipitation in application, and hence confirmed punicalin to be a promising antioxidant.

Cumulation of Tabernanthe Iboga alkaloid and its metabolite in organs of mice

The accumulation of Tabernanthe Iboga alkaloid ibogaine and its metabolite noribogaine in liver, kidney, spleen, heart, brain and muscle after administration directly into the stomach in white laboratory mice were studied. For this purpose a high-performance liquid chromatography (HPLC) method with fluorescein as internal standart were chosen. Solid phase extraction (SPE) method was employed to isolate the tested substances from the internal organs. Separation of the analytes was performed on a reversed-phase XTerra RP18 analytical column (3×150 mm, particle size 3.5 µm) using a gradient, the mobile phase consisted of a mixture acetonitril and 0.1% TFA in water. The total run cycle was 25 min. The flow rate was 0.4 ml/min, and injection volume was 10 µl. The excitation wavelength was set at 230 nm and emission wavelength at 336 nm. Excitation wavelength of internal standard (fluoresceine) was set at 440 nm and emission wavelength at 514 nm. Experiment demonstrated that highest concentration of ibogaine and noribogaine have been obtained in spleen and the lower one – in muscle of the animal after the direct administration into the mice stomach. Key words: Tabernanthe Iboga, ibogaine, noribogaine, solid phase extraction, distribution in mice organs, HPLC.

Quantitative Assessment of Chromatin Immunoprecipitation Grade Antibodies Directed against Histone Modifications Reveals Patterns of Co-occurring Marks on Histone Protein Molecules

The defining step in most chromatin immunoprecipitation (ChIP) assays is the use of an antibody to enrich for a particular protein or histone modification state associated with segments of chromatin. The specificity of the antibody is critical to the interpretation of the experiment, yet this property is rarely reported. Here, we present a quantitative method using mass spectrometry to characterize the specificity of key histone H3 modification-targeting antibodies that have previously been used to characterize the "histone code." We further extend the use of these antibody reagents to the observation of long range correlations among disparate histone modifications. Using purified human histones representing the mixture of chromatin states present in living cells, we were able to quantify the degree of target enrichment and the specificity of several commonly used, commercially available ChIP grade antibodies. We found significant differences in enrichment efficiency among various reagents directed against four frequently studied chromatin marks: H3K4me2, H3K4me3, H3K9me3, and H3K27me3. For some antibodies, we also detected significant off target enrichment of alternate modifications at the same site (i.e., enrichment of H3K4me2 by an antibody directed against H3K4me3). Through cluster analysis, we were able to recognize patterns of co-enrichment of marks at different sites on the same histone protein. Surprisingly, these co-enrichments corresponded well to "canonical" chromatin states that are exemplary of activated and repressed regions of chromatin. Altogether, our findings suggest that 1) the results of ChIP experiments need to be evaluated with caution given the potential for cross-reactivity of the commonly used histone modification recognizing antibodies, 2) multiple marks with consistent biological interpretation exist on the same histone protein molecule, and 3) some components of the histone code may be transduced on single proteins in living cells.

Characterization of hydroxypropylmethylcellulose (HPMC) using comprehensive two-dimensional liquid chromatography

Various hydroxyl-propylmethylcellulose (HPMC) polymers were characterized according to size and compositional distributions (percentage of methoxyl and hydroxyl-propoxyl substitution) by means of comprehensive two-dimensional liquid chromatography (LC × LC) using reversed-phase (RP) liquid chromatography in the first dimension and aqueous size-exclusion chromatography (aq-SEC) in the second dimension. RP separation was carried out in gradient-elution mode applying 0.05% TFA in water and 1-propanol, while 0.05% TFA in water was used as mobile phase in aqueous SEC. A two-position ten-port switching valve equipped with two storage loops was used to realize LC × LC. Detection of HPMC was accomplished by charged-aerosol detection (CAD). Data processing to visualize chromatograms was carried out using Matlab software. The significant influence of the LC × LC temperature on (the retention of) HPMC was studied using a column oven which allowed accurate temperature control. Due to the phenomenon of thermal gelation, which is a result of methyl and hydroxypropyl substitution of anhydroglucose units from the cellulose backbone, we were able to obtain additional, specific information on compositional characteristics of various HPMC samples. As the retention behaviour of gelated and non-gelated polymer proved to be different, the fraction of the polymer that is gelated in the chromatographic column could be monitored at different temperatures. Moreover, the temperature at which half of the polymer is gelated could be correlated with the cloud-point temperature. As a result, differences in inherent cloud points of modified cellulose can be used as a further distinguishing property in “temperature-responsive” LC × LC.

Abstract LB-463: Proteomic patterns of colonic submucosa delineates the inflammatory colitides. This could aid understand IBD-related colorectal malignancy

Abstract Purpose: Differentiating Crohn's colitis (CC) and ulcerative colitis (UC) can be challenging even in combination of clinical, endoscopic, radiologic and histopathology examination. Biomarker studies have thus far been unsuccessful for disease delineation. We aim to use unique tissue proteomic methods to evaluate colonic tissue layers for potential biomarkers to identify CC vs. UC. Methods: Fresh-frozen colon specimens from resections for IBD and/or colorectal cancer were retrospectively retrieved. Colitis diagnoses were histologically re-confirmed by a blinded gastrointestinal pathologist. Three sample groups (n=5 each group) were examined: normal colon from CRC specimens (control), UC & CC. MALDI-MS was used to profile mucosal and submucosal compartments individually. Frozen tissues were sectioned at ∼10–15 μm for mounting onto either metal or conductive glass target plates (the glass plates allowing for histologic and MALDI-MS analysis on the same section). Sinapinic acid (20 mg/mL in 50:50 acetonitrile: 0.1% TFA in water) was used to give the best combination of uniform crystal coverage and signal quality for direct tissue protein analysis. Results: MALDI-MS achieved high mass accuracy (±0.01 Daltons) in the lower mass range (<15 kDa). There was distinguishable isotopic resolution of mass-to-charge ratio (m/z) values between normal vs the IBD and more strikingly between CC vs. UC (p< 0.0009) of SAM and FDR tests. There were 5 statistically significant discriminative m/z peaks observed in CC vs UC submucosa. The mucosa did not yield such distinctive peaks. Both the mucosa and submucosa provided signatures that differentiated IBD (CC or UC)) from control tissues (p< 0.001). Conclusions: MALDI-MS tissue profiling as described distinguished the colitides. The methodology revealed 5 m/z peaks of interest. Analyses are underway to identify these IBD discriminative proteins. We hypothesize that these biomarker candidates that distinguish UC from CC will allow delineation of indeterminate colitis into UC or CC. Whether these protein fingerprints play a role in colonic carcinogenesis remains to be elucidated. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-463. doi:10.1158/1538-7445.AM2011-LB-463

Preparative separation of punicalin from waste water of hydrolysed pomegranate husk by macroporous resin and preparative high-performance liquid chromatography

The present study investigated a fast and cost-effective method for the preparation of punicalin from waste water of hydrolysed pomegranate husk by H-103 macroporous resin and preparative high-performance liquid chromatography (HPLC) using a mobile phase consisting of 10% (v/v) of methanol and 0.1% TFA in water for primary separation and a mobile phase consisting of 30% (v/v) of methanol and 0.1% TFA in water to remove gallic acid and other minor impurities. 120ml of waste water, which contained about 480mg punicalin, could produce 1147mg of punicalin-rich extract of 36.4% punicalin after separation by H-103 macroporous resin. 349mg of 97% punicalin could be obtained after purified by preparative HPLC. The recovery per litre of waste water was more than 70%. The product was assessed by analytical HPLC and characterised by MS and 1H NMR and 13C NMR. This method is practical for industrial applications and could provide a low-cost means to use a currently underutilized industrial by-product.

Determination of Major Bovine Milk Proteins by Reversed Phase High Performance Liquid Chromatography

Abstract A reversed-phase high-performance liquid chromatography (RP-HPLC) for the quantification of six major bovine milk proteins (κ-casein (κ-CN), αs2-casein (αs2-CN), αs1-casein (αs1-CN), β-casein (β-CN), immunoglobulins (IgG) and whey) was developed. The separation and quantification of these major proteomic components were achieved with an Agilent Zorbax 300SB-C8 analytical column (250 mm × 4.6 mm I.D., 5 μm) at 45 °C with detection wavelength at 214 nm. The gradient elution was carried out with solvents of 0.1% TFA in water and 0.1% TFA in acetonitrile at a flow rate of 0.8 ml min−1. The method validation consisted of testing the linearity, repeatability, reproducibility and accuracy. The recoveries of six target proteins spiked in milk were in the range from 74.8% to 132.5%. Nine different commercial milk brands were used, the difference of the concentration and relative ration of κ-CN, αs2-CN, αs1-CN, β-CN and whey were found.

Simultaneous Determination of Major Constituents of Honeybee Venom by LC-DAD

The aim of the study was to develop an LC method for honeybee venom analysis, using cytochrome c as an internal standard. The SynChropack C8 6.5 μm, 4.6 × 100 mm column was applied. The bee venom was separated by linear gradient 5–80% B at 30 min (eluent A—0.1% TFA in water, eluent B—0.1% TFA in acetonitrile:water (80:20)). The flow rate of mobile phase was maintained at 1 mL min−1, injection volume: 40 μL, separation temperature: 25 °C. The analysis was monitored at 220 nm. Several honeybee venom constituents were separated and the content of four of them (apamine, mast cell degranulating peptide, phospholipase A2 and melittin) were determined. By applying this methodology differences in chemical composition of honeybee venom were evaluated. In order to confirm the data obtained, the following steps and parameters were taken into account for the validation of the method: selectivity, precision (injection repeatability, analysis repeatability), accuracy (recovery), linearity and operating range, limit of detection and limit of quantitation. All steps of validation proved that the developed analytical procedure was suitable for its intended purpose (standardization). Due to its simplicity, the developed method can be easily automated and incorporated into routine operations both in the bee venom identification, quality control and assay tests.

The Impact of Insulin Adsorption onto the Infusion Sets in the Adult Intensive Care Unit

The difficulty in achieving tight glycemic control (TGC)1,2 in the adult intensive care unit (ICU) is well documented.3,4 We aimed to explore the impact of insulin adsorption onto infusion sets in the laboratory and to assess retrospectively if higher insulin doses are required after syringe changes in practice in the ICU. Adsorption is reported to occur predominantly during the first hour of infusion, after which binding sites are saturated.5 There is greater insulin recovery with faster flow rates and higher insulin concentration.6 The clinical significance of such adsorption in the ICU setting is uncertain.5 In our study—approved by the local ethics committee—adsorption of insulin onto the giving sets used in our adult ICU was analyzed. The infusion set used was a 50 ml polypropylene syringe (BD Plastipak®) and 1.6 ml/200 cm polyethylene (PE) tubing (Cardinal Health, extension set). In the laboratory, the concentration investigated was 1 U/ml of neutral insulin in sodium chloride 0.9%. The concentrations were determined using a high-performance liquid chromatography method with a Jupitor 300, 5 μm C18, 250 mm × 4.6 mm column (Phenomenex), a mobile phase of 30% acetonitrile and 0.1% TFA in water, and ultraviolet detection at 210 nm. Significant insulin adsorption of 10% occurred during the first hour of an infusion when the infusion rate was 1 ml/h (Figure 1). Our experience suggests that this is a common infusion rate used in intensive insulin therapy. No significant adsorption occurred when the infusion rate was 4 ml/h, although a trend could be seen with solution leaving the PE tubing at the beginning of the time period having a lower insulin concentration, which increased with time. Figure 1. The mean (± standard error) insulin concentration versus time with an infusion rate of 1 U/h. The infusion rates used before and after an infusion set change were investigated for 17 ICU patients known to have been managed with a TGC protocol, aiming for glycemia of 4.4–6.1 mmol/liter, to examine any relationship between the duration of usage of the infusion set and the variation in insulin dosing required. These patients were not prescribed drugs known to affect glucose control, such as inotropes and corticosteroids. In the ICU patients, a significantly higher insulin infusion rate was required after an infusion set change when the infusion rate was 1 ml/h or lower. After an infusion set change, the rate was adjusted upward in 76.5% of cases. In conclusion, we found in the laboratory that less insulin is delivered during the first three hours after an infusion set change, when the infusion rate is 1 ml/h or lower, due to adsorption onto infusion sets. In the ICU, a higher infusion rate was frequently required after an infusion set change when the infusion rate was 1 ml/h or lower. Our results indicate that at low infusion rates, if the syringe and line are not primed, only 90% expected insulin is delivered. The clinical impact of a loss of 0.1 U/h of insulin is questionable, but in practice when syringes were changed, higher rates were frequently required. Further investigation should be undertaken to confirm these findings.

Enhanced Detection of Low Abundance Human Plasma Proteins Using a Tandem IgY12-SuperMix Immunoaffinity Separation Strategy

The enormous dynamic range of human bodily fluid proteomes poses a significant challenge for current MS-based proteomics technologies as it makes it especially difficult to detect low abundance proteins in human biofluids such as blood plasma, which is an essential aspect for successful biomarker discovery efforts. Here we present a novel tandem IgY12-SuperMix immunoaffinity separation system for enhanced detection of low abundance proteins in human plasma. The tandem IgY12-SuperMix system separates approximately 60 abundant proteins from the low abundance proteins in plasma, allowing for significant enrichment of low abundance plasma proteins in the SuperMix flow-through fraction. High reproducibility of the tandem separations was observed in terms of both sample processing recovery and LC-MS/MS identification results based on spectral count data. The ability to quantitatively measure differential protein abundances following application of the tandem separations was demonstrated by spiking six non-human standard proteins at three different levels into plasma. A side-by-side comparison between the SuperMix flow-through and IgY12 flow-through samples analyzed by both one- and two-dimensional LC-MS/MS revealed a 60-80% increase in proteome coverage as a result of the SuperMix separations, suggesting significantly enhanced detection of low abundance proteins. A total of 695 plasma proteins were confidently identified in a single analysis (with a minimum of two peptides per protein) by coupling the tandem separation strategy with two-dimensional LC-MS/MS, including 42 proteins with reported normal concentrations of approximately 100 pg/ml to 100 ng/ml. The concentrations of two selected proteins, macrophage colony-stimulating factor 1 and matrix metalloproteinase-8, were independently validated by ELISA as 202 pg/ml and 12.4 ng/ml, respectively. Evaluation of binding efficiency revealed that 45 medium abundance proteins were efficiently captured by the SuperMix column with >90% retention. Taken together, these results illustrate the potential broad utilities of this tandem IgY12-SuperMix strategy for proteomics applications involving human biofluids where effectively addressing the dynamic range challenge of the specimen is imperative.

Analysis of trifluoroacetic acid and other short-chain perfluorinated acids (C2–C4) in precipitation by liquid chromatography–tandem mass spectrometry: Comparison to patterns of long-chain perfluorinated acids (C5–C18)

A method has been developed to measure 29 perfluorinated acids (PFAs) including short-chain perfluorocarboxylates (PFCAs) such as trifluoroacetic acid (TFA; C2) and long-chain PFCAs, perfluoroalkylsulfonates, fluorotelomer acids, and two perfluorooctylsulfonamides in water matrices. The method involves solid phase extraction (SPE) using a weak anion-exchange (WAX) cartridge, an ion-exchange high-performance liquid chromatography (HPLC) column separation, and tandem mass spectrometry (MS/MS) detection. To our knowledge, this is the first HPLC–MS/MS method to determine TFA in water at sub-ng L −1 concentrations. The method is selective, simple, and robust, capable of measuring 29 PFAs in a single analysis, with overall recoveries of the target analytes ranging from 75% to 132%. The method was applied to the analysis of rainwater samples collected from two cities in Japan. TFA and several short-chain PFAs were the major compounds found in rainwater.

Identification of Glioma Neovascularization-related Proteins by Using MALDI-FTMS and Nano-LC Fractionation to Microdissected Tumor Vessels

The identification of angiogenesis-related proteins is important for the development of new antiangiogenic therapies, and such proteins are potential new biomarkers for gliomas. The aim of this study was to identify proteins that are exclusively present in glioma neovasculature and not in the vasculature of normal brain. We combined advanced proteomics techniques to compare the expression profiles of microdissected blood vessels from glioma with blood vessels of normal control brain samples. We measured the enzymatic generated peptide profiles from these microdissected samples by MALDI-FTMS. Subsequently, the samples were fractionated by nano-LC prior to MALDI-TOF/TOF. This combined approach enabled us to identify four proteins that appeared to be exclusively expressed in the glioma blood vessels. Two of these proteins, fibronectin and colligin 2, were validated on tissue sections using specific antibodies. We found that both proteins are present in active angiogenesis in glioma, other neoplasms, and reactive conditions in which neoangiogenesis takes place. This work proves that gel-free mass spectrometric techniques can be used on relatively small numbers of cells generated by microdissection procedures to successfully identify differentially expressed proteins.

Development and Validation of an HPLC/UV Method for Quantification of Bioactive Peptides in Fermented Milks

The simultaneous separation and quantification of two casein peptides (IPP, VPP) presenting potent inhibitory activity of angiotensin‐converting‐enzyme (ACE) and casein in fermented milks was developed. Gradient elution was carried out at a flow‐rate of 1 mL/min, using a mixture of two solvents. Solvent A was 0.1% TFA in water and solvent B was acetonitrile‐water‐trifluoracetic acid 95∶5∶0.1. The effluent was monitored by UV detector at 214 nm. Calibration curves were constructed in the interval of 0.01–1.0 mg/mL for VPP, 0.005–1.0 mg/mL for IPP, and 0.05–3.0 mg/mL for casein. R2 invariably exceeded 0.999. The detection limits were 0.004 for VPP, 0.002 mg/mL for IPP, and 0.02 mg/mL for casein. Repeatability of the method was evaluated by six consecutive injections of two standard solutions containing VPP, IPP, and casein. The RSD values for concentration were all below 5.08%. Recovery studies were carried out to determine the accuracy of the method. Recoveries ranged between 88 and 98.2%. The methodology was applied, not only, for the monitorization of VPP, IPP, and casein in commercial fermented milks labeled as presenting antihypertensive properties, but also, in milk with different degrees of fermentation by L. Helveticus, and in other commercial functional fermented milks, such as, those presenting cholesterol lowering properties.

Synthesis and evaluation of 2-(2-fluoro-4-hydroxymethyl-5-methoxy-phenoxy)acetic acid as a linker in solid-phase synthesis monitored by gel-phase19F NMR spectroscopy

Gel-phase (19)F NMR spectroscopy is a useful monitoring technique for solid-phase organic chemistry due to the high information content it delivers and swift acquisition times, using standard NMR spectrometers. This paper describes the synthesis of the novel linker 2-(2-fluoro-4-hydroxymethyl-5-methoxy-phenoxy)acetic acid in 29% yield over seven steps, using nucleophilic aromatic substitutions on 2,4,5-trifluorobenzonitrile as key steps. Following standard solid-phase synthesis a peptide could be cleaved from the linker using 20% TFA in CH(2)Cl(2) in 30 minutes, in contrast to a previously described monoalkoxy linker that requires 90% TFA in water at elevated temperature. A resin-bound peptide could be successfully glycosylated using only two equivalents of a thioglycoside donor, activated with N-iodosuccinimide and trifluoromethanesulfonic acid, and subsequent cleavage and deprotection gave the target glycopeptide. Direct glycosylation of the linker itself followed by mild acidic cleavage gave a fully protected hemiacetal for further chemical manipulation.

Analysis of the Salmonella typhimurium Proteome through Environmental Response toward Infectious Conditions

Salmonella enterica serovar Typhimurium (also known as Salmonella typhimurium) is a facultative intracellular pathogen that causes approximately 8,000 reported cases of acute gastroenteritis and diarrhea each year in the United States. Although many successful physiological, biochemical, and genetic approaches have been taken to determine the key virulence determinants encoded by this organism, the sheer number of uncharacterized reading frames observed within the S. enterica genome suggests that many more virulence factors remain to be discovered. We used a liquid chromatography-mass spectrometry-based "bottom-up" proteomic approach to generate a more complete picture of the gene products that S. typhimurium synthesizes under typical laboratory conditions as well as in culture media that are known to induce expression of virulence genes. When grown to logarithmic phase in rich medium, S. typhimurium is known to express many genes that are required for invasion of epithelial cells. Conversely stationary phase cultures of S. typhimurium express genes that are needed for both systemic infection and growth within infected macrophages. Lastly bacteria grown in an acidic, magnesium-depleted minimal medium (MgM) designed to mimic the phagocytic vacuole have been shown to up-regulate virulence gene expression. Initial comparisons of protein abundances from bacteria grown under each of these conditions indicated that the majority of proteins do not change significantly. However, we observed subsets of proteins whose expression was largely restricted to one of the three culture conditions. For example, cells grown in MgM had a higher abundance of Mg(2+) transport proteins than found in other growth conditions. A second more virulent S. typhimurium strain (14028) was also cultured under these same growth conditions, and the results were directly compared with those obtained for strain LT2. This comparison offered a unique opportunity to contrast protein populations in these closely related bacteria. Among a number of proteins displaying a higher abundance in strain 14028 were the products of the pdu operon, which encodes enzymes required for propanediol utilization. These pdu operon proteins were validated in culture and during macrophage infection. Our work provides further support for earlier observations that suggest pdu gene expression contributes to S. typhimurium pathogenesis.

A Chemical Proteomics Approach for the Identification of Accessible Antigens Expressed in Human Kidney Cancer

A promising avenue toward the development of more selective anticancer drugs consists in the targeted delivery of bioactive molecules to the tumor environment by means of binding molecules specific to tumor-associated markers. We have used a chemical proteomics approach based on the ex vivo perfusion and biotinylation of accessible structures within surgically resected human kidneys with tumor to gain information about accessible and abundant antigens that are overexpressed in human cancer. This procedure led to the selective labeling with biotin of vascular structures. Biotinylated proteins were purified on streptavidin resin and identified using mass spectrometric methodologies, revealing 637 proteins, 184 of which were only found in tumor specimens and 223 of which were only found in portions of normal kidneys. Immunohistochemical and PCR analysis confirmed that several of the putative cancer antigens identified in this study are indeed preferentially expressed in tumors. In conclusion, we have developed a methodology that allows the identification of accessible biomarkers in human tissues. The tumor-associated antigens identified in this study may be suitable targets for antibody-based anticancer therapies. The experimental approach described here should be applicable to other surgical specimens and to other pathologies as well as to the study of basic physiological and immunological processes.

Evaluation of Multiprotein Immunoaffinity Subtraction for Plasma Proteomics and Candidate Biomarker Discovery Using Mass Spectrometry

Strategies for removal of high abundance proteins have been increasingly utilized in proteomic studies of serum/plasma and other body fluids to enhance the detection of low abundance proteins and achieve broader proteome coverage; however, both the reproducibility and specificity of the high abundance protein depletion process still represent common concerns. Here we report a detailed evaluation of immunoaffinity subtraction performed applying the ProteomeLab IgY-12 system that is commonly used in human serum/plasma proteome characterization in combination with high resolution LC-MS/MS. Plasma samples were repeatedly processed using this approach, and the resulting flow-through fractions and bound fractions were individually analyzed for comparison. The removal of target proteins by the immunoaffinity subtraction system and the overall process was highly reproducible. Non-target proteins, including one spiked protein standard (rabbit glyceraldehyde-3-phosphate dehydrogenase), were also observed to bind to the column at different levels but also in a reproducible manner. The results suggest that multiprotein immunoaffinity subtraction systems can be readily integrated into quantitative strategies to enhance detection of low abundance proteins in biomarker discovery studies.

Differential Label-free Quantitative Proteomic Analysis of Shewanella oneidensis Cultured under Aerobic and Suboxic Conditions by Accurate Mass and Time Tag Approach

We describe the application of LC-MS without the use of stable isotope labeling for differential quantitative proteomic analysis of whole cell lysates of Shewanella oneidensis MR-1 cultured under aerobic and suboxic conditions. LC-MS/MS was used to initially identify peptide sequences, and LC-FTICR was used to confirm these identifications as well as measure relative peptide abundances. 2343 peptides covering 668 proteins were identified with high confidence and quantified. Among these proteins, a subset of 56 changed significantly using statistical approaches such as statistical analysis of microarrays, whereas another subset of 56 that were annotated as performing housekeeping functions remained essentially unchanged in relative abundance. Numerous proteins involved in anaerobic energy metabolism exhibited up to a 10-fold increase in relative abundance when S. oneidensis was transitioned from aerobic to suboxic conditions.

Analysis of phenolic glycosides and saponins in Primulaelatior and Primulaveris (primula root) by liquid chromatography, evaporative light scattering detection and mass spectrometry

This paper describes the first liquid chromatographic method suitable for the simultaneous determination of bioactive compounds, saponins and phenolic glycosides, present in Primula elatior and Primula veris, including the NMR data of primulaverin and primeverin. Optimum separations were obtained with a Synergi 4 μm Fusion RP 80 Å column, using 0.025% TFA in water and 5% acetonitrile in methanol as mobile phase. Saponins were detected by evaporative light scattering detection (ELSD), whereas the phenolic glycosides were monitored by UV at 210 nm. The method was validated for repeatability ( σ rel ≤ 4.5%), precision (intra- and inter-day variation ≤5.0%), accuracy (recovery ≥97.1%) and sensitivity (LOD ≤22 ng (UV) and ≤38 ng (ELSD) on-column, respectively). LC–MS experiments in negative APCI mode allowed a final peak assignment. Both Primula species could easily be differentiated by their saponin pattern. The total saponin content was highest in P. veris roots (max. 14.9%), the aerial parts or P. elatior contained significantly less amounts; primeverin (0.64–1.42%) showed to be the most dominant phenolic glycoside.