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  • LC/MS/MS structure elucidation of reaction intermediates formed during the TiO2 photocatalysis of microcystin-LR 18377943

    Microcystin-LR (MC-LR), a cyanotoxin and emerging drinking water contaminant, was treated with TiO2 photocatalysts immobilized on stainless steel plates as an alternative to nanoparticles in slurry. The reaction intermediates of MC-LR were identified with mass spectrometry (MS) at pH of Milli-Q water (pHsq = 5.7). Eleven new [M+H]+ were observed in the liquid chromatography mass spectrometry (LC/MS) chromatogram with some of them giving multiple peaks. Most of these reaction intermediates have not been reported from previous studies employing TiO2 nanoparticles at acidic conditions (pH = 4.0). Investigating the effects of pH (for 3.0ms showed that acidic conditions are preferable for the degradation. Combined with the limited surface area of the films and the absence of additional oxidants (i.e., H2O2) the degradation was slower and more intermediate steps were identified. Possible structures of the intermediates (formed at neutral pH) after analyzing the corresponding MS/MS spectra are reported. The collision-induced dissociation of the [M+H]+ of MC-LR and the intermediates 1011.5 and 1029.5 are discussed and possible fragmentation pathways and mechanisms are also proposed. Analysis of the MS/MS spectra indicates that the fragmentation of some amino acids is less favorable because of internal interaction with free groups of adjacent amino acids. The MS/MS spectra assisted in determining hydroxylation sites, by the formation or alteration of specific product ions such as m/z 599.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple

  • Large-scale quantitative LC-MS/MS analysis of detergent-resistant membrane proteins from rat renal collecting duct. 18596208

    In the renal collecting duct, vasopressin controls transport of water and solutes via regulation of membrane transporters such as aquaporin-2 (AQP2) and the epithelial urea transporter UT-A. To discover proteins potentially involved in vasopressin action in rat kidney collecting ducts, we enriched membrane "raft" proteins by harvesting detergent-resistant membranes (DRMs) of the inner medullary collecting duct (IMCD) cells. Proteins were identified and quantified with LC-MS/MS. A total of 814 proteins were identified in the DRM fractions. Of these, 186, including several characteristic raft proteins, were enriched in the DRMs. Immunoblotting confirmed DRM enrichment of representative proteins. Immunofluorescence confocal microscopy of rat IMCDs with antibodies to DRM proteins demonstrated heterogeneity of raft subdomains: MAL2 (apical region), RalA (predominant basolateral labeling), caveolin-2 (punctate labeling distributed throughout the cells), and flotillin-1 (discrete labeling of large intracellular structures). The DRM proteome included GPI-anchored, doubly acylated, singly acylated, cholesterol-binding, and integral membrane proteins (IMPs). The IMPs were, on average, much smaller and more hydrophobic than IMPs identified in non-DRM-enriched IMCD. The content of serine 256-phosphorylated AQP2 was greater in DRM than in non-DRM fractions. Vasopressin did not change the DRM-to-non-DRM ratio of most proteins, whether quantified by tandem mass spectrometry (LC-MS/MS, n=22) or immunoblotting (n=6). However, Rab7 and annexin-2 showed small increases in the DRM fraction in response to vasopressin. In accord with the long-term goal of creating a systems-level analysis of transport regulation, this study has identified a large number of membrane-associated proteins expressed in the IMCD that have potential roles in vasopressin action.
    Document Type:
    Reference
    Product Catalog Number:
    05-184

  • GEL-FREE SAMPLE PREPARATION FOR THE NANOSCALE LC-MS/MS ANALYSIS AND IDENTIFICATION OF LOW-NANOGRAM PROTEIN SAMPLES 17763504

    Protein identification at the low nanogram level could in principle be obtained by most nanoscale LC-MS/MS systems. Nevertheless, the complex sample preparation procedures generally required in biological applications, and the consequent high risk of sample losses, very often hamper practical achievement of such low levels. In fact, the minimal amount of protein required for the identification from a gel band or spot, in general, largely exceeds the theoretical limit of identification reachable by nanoscale LC-MS/MS systems. A method for the identification of low levels of purified proteins, allowing limits of identification down to 1 ng when using standard bore, 75 microm id nanoscale LC-MS/MS systems is here reported. The method comprises an offline two-step sample cleanup, subsequent to protein digestion, which is designed to minimize sample losses, allows high flexibility in the choice of digestion conditions and delivers a highly purified peptide mixture even from "real world" digestion conditions, thus allowing the subsequent nanoscale LC-MS/MS analysis to be performed in automated, unattended operation for long series. The method can be applied to the characterization of low levels of affinity purified protei
    Document Type:
    Reference
    Product Catalog Number:
    C5737
    Product Catalog Name:
    ZipTip® Pipette Tips

  • Evaluation of HPLC reagent water purity via LC-MS and total organic carbon analysis Evaluation of HPLC reagent water purity via LC-MS and total organic carbon analysis

    An important factor in optimizing LC-MS analysis is the use of solvents and chemical reagents of high purity. When converting from HPLC with UV detection to LCMS, the purity of water used for the mobile phase becomes critical. In addition to column blinding, ghost peaks, and other problems caused by excess organics in HPLC, organic contamination creates high background and causes a loss of sensitivity in LC-MS. Although confirmation via MS-MS, for example, would be required for precise identification of species detected in the water,2 the presence and magnitude of the selected spectra reasonably indicate the relative purity of these waters. This was particularly visible when using bottled HPLC-reagent waters. Analysis of HPLC-grade water using UV detection at 214 and 254 nm is not a suitable quality control for LC-MS applications. On-line measurement of organics as TOC appears to provide a rapid and timely indication of the organic purity of water suitable to successfully perform LC-MS. Additionally, ion exchange combined with UV photooxidation offers a benefit to LC-MS users over conventional water systems that use ion-exchange media alone. The observations and methods used may provide a screening method to monitor the quality of reagent waters used in HPLC and LC-MS in order to obtain optimal results.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Benefits of the pretreatment step in purifying water for LC-MS analyses Benefits of the pretreatment step in purifying water for LC-MS analyses

    Solvent and reagent quality has long been a topic of interest to analytical chemists using liquid chromatography. While several articles describe chromatography methods, few references address the purity of solvents used to prepare mobile phases. Some data to support the water quality suitable for HPLC and LC-MS analysis have been presented previously, but little has been published on the means required to achieve such water quality. Starting from a customer case study, data reported here show the benefits of optimizing each step of the water purification process. Indeed, water purification can be divided into two major basic steps, the pre-treatment step and the polishing step. Since water delivered at the final purification stage is used to prepare the mobile phase,it seems to be an obvious target for optimization., However, the initial pretreatment step is equally critical. Several pretreatment technologies are discussed for their ability and suitability to be utilized in complete water purification processes dedicated to produce water for HPLC and LC-MS work.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • FOOD PEPTIDOMICS OF IN VITRO GASTROINTESTINAL DIGESTIONS OF PARTIALLY PURIFIED BOVINE HEMOGLOBIN: LOW-RESOLUTION VERSUS HIGH-RESOLUTION LC-MS/MS ANALYSES 26990205

    Consumers and governments have become aware how the daily diet may affect the human health. All proteins from both plant and animal origins are potential sources of a wide range of bioactive peptides and the large majority of those display health-promoting effects. In the meat production food chain, the slaughterhouse blood is an inevitable co-product and, today, the blood proteins remain underexploited despite their bioactive potentiality. Through a comparative food peptidomics approach we illustrate the impact of resolving power, accuracy, sensitivity, and acquisition speed of low-resolution (LR)- and high-resolution (HR)-LC-ESI-MS/MS on the obtained peptide mappings and discuss the limitations of MS-based peptidomics. From in vitro gastrointestinal digestions of partially purified bovine hemoglobin, we have established the peptide maps of each hemoglobin chain. LR technique (normal bore C18 LC-LR-ESI-MS/MS) allows us to identify without ambiguity 75 unique peptides while the HR approach (nano bore C18 LC-HR-ESI-MS/MS) unambiguously identify more than 950 unique peptides (post-translational modifications included). Herein, the food peptidomics approach using the most performant separation methods and mass spectrometers with high-resolution capabilities appears as a promising source of information to assess the health potentiality of proteins.
    Document Type:
    Reference
    Product Catalog Number:
    C5737
    Product Catalog Name:
    ZipTip® Pipette Tips

  • High-throughput identification of IMCD proteins using LC-MS/MS. 16449382

    The inner medullary collecting duct (IMCD) is an important site of vasopressin-regulated water and urea transport. Here we have used protein mass spectrometry to investigate the proteome of the IMCD cell and how it is altered in response to long-term vasopressin administration in rats. IMCDs were isolated from inner medullas of rats, and IMCD proteins were identified by liquid chromatography/tandem mass spectrometry (LC-MS/MS). We present a WWW-based "IMCD Proteome Database" containing all IMCD proteins identified in this study (n = 704) and prior MS-based identification studies (n = 301). We used the isotope-coded affinity tag (ICAT) technique to identify IMCD proteins that change in abundance in response to vasopressin. Vasopressin analog (dDAVP) or vehicle was infused subcutaneously in Brattleboro rats for 3 days, and IMCDs were isolated for proteomic analysis. dDAVP and control samples were labeled with different cleavable ICAT reagents (mass difference 9 amu) and mixed. This was followed by one-dimensional SDS-PAGE separation, in-gel trypsin digestion, biotin-avidin affinity purification, and LC-MS/MS identification and quantification. Responses to vasopressin for a total of 165 proteins were quantified. Quantification, based on semiquantitative immunoblotting of 16 proteins for which antibodies were available, showed a high degree of correlation with ICAT results. In addition to aquaporin-2 and gamma-epithelial Na channel (gamma-ENaC), five of the immunoblotted proteins were substantially altered in abundance in response to dDAVP, viz., syntaxin-7, Rap1, GAPDH, heat shock protein (HSP)70, and cathepsin D. A 28-protein vasopressin signaling network was constructed using literature-based network analysis software focusing on the newly identified proteins, providing several new hypotheses for future studies.
    Document Type:
    Reference
    Product Catalog Number:
    06-471

  • Analysis of modified apolipoprotein B-100 structures formed in oxidized low-density lipoprotein using LC-MS/MS. 17549798

    Oxidatively modified low-density lipoprotein (oxLDL) is one of the major factors involved in the development of atherosclerosis. Because of the insolubility of apolipoprotein B-100 (apoB-100) and the heterogeneous nature of oxidative modification, modified structures of apoB-100 in oxLDL are poorly understood. We applied an on-Membrane sample preparation procedure for LC-MS/MS analysis of apoB-100 proteins in native and modified low-density lipoprotein (LDL) samples to eliminate lipid components in the LDLs followed by collection of tryptic digests of apoB-100. Compared with a commonly used in-gel digestion protocol, the sample preparation procedure using PVDF membrane greatly increased the recovery of tryptic peptides and resulted in improved sequence coverage in the final analysis, which lead to the identification of modified amino acid residues in copper-induced oxLDL. A histidine residue modified by 4-hydroxynonenal, a major lipid peroxidation product, as well as oxidized histidine and tryptophan residues were detected. LC-MS/MS in combination with the on-Membrane sample preparation procedure is a useful method to analyze highly hydrophobic proteins such as apoB-100.
    Document Type:
    Reference
    Product Catalog Number:
    C5737
    Product Catalog Name:
    ZipTip® Pipette Tips