Dithranol as a MALDI Matrix for Tissue Imaging of Lipids by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

To fill the unmet need for improved matrixes for matrix-assisted laser desorption ionization (MALDI) tissue imaging of small molecules, dithranol (DT)a matrix mainly used for the analysis of synthetic polymerswas evaluated for detection of lipids in rat liver and bovine calf lens, using MALDI Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS). The use of DT resulted in better detection of endogenous lipids than did two other commonly used matrixes, α-cyano-4-hydroxycinnamic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB), with >70 lipid entities (including phosphatidylcholines, phosphatidylethanolamines, sphingomyelins, phosphatidylserines, phosphatidylglycerol, phosphatidic acids, ceramide phosphates, sterol lipids, acyl carnitines, and glycerides) being detected in rat liver and bovine lens tissue sections, using positive-ion detection. Using saturated DT in chloroform/methanol (2:1, v/v), with 1% formic acid in the final mixture, 57 lipid entities were successfully imaged from bovine calf lens, with clear and distinct distribution patterns. In a section across the lens equatorial plane, all compounds showed concentric distributions around the lens nucleus and most showed specific abundance changes, which correlated with lens fiber cell age. As a novel finding, palmitoylcarnitine and oleoylcarnitine were found uniquely localized to the younger lens fiber cell cortex region. This work demonstrates the potential of DT as a new matrix for tissue imaging by MALDI-FTICR MS

Proteomic Profiling of <i>Leishmania donovani</i> Promastigote Subcellular Organelles

To facilitate a greater understanding of the biological processes in the medically important <i>Leishmania donovani</i> parasite, a combination of differential and density-gradient ultracentrifugation techniques were used to achieve a comprehensive subcellular fractionation of the promastigote stage. An in-depth label-free proteomic LC–MS/MS analysis of the density gradients resulted in the identification of ∼50% of the <i>Leishmania</i> proteome (3883 proteins detected), which included ∼645 integral membrane proteins and 1737 uncharacterized proteins. Clustering and subcellular localization of proteins was based on a subset of training <i>Leishmania</i> proteins with known subcellular localizations that had been determined using biochemical, confocal microscopy, or immunoelectron microscopy approaches. This subcellular map will be a valuable resource that will help dissect the cell biology and metabolic processes associated with specific organelles of <i>Leishmania</i> and related kinetoplastids

Subzero Temperature Chromatography and Top-Down Mass Spectrometry for Protein Higher-Order Structure Characterization: Method Validation and Application to Therapeutic Antibodies

Characterization of the higher-order structure and structural dynamics of proteins is crucial for in-depth understanding of their functions. Amide hydrogen/deuterium exchange (HDX), monitored by mass spectrometry (MS), is now a popular technique for measuring protein higher-order structural changes. Although the proteolysis-based HDX-MS approach is most commonly used, the “top-down” approach, which fragments intact proteins directly using electron-based dissociation, is becoming an important alternative and has several advantages. However, the commonly used top-down strategies are direct-infusion based and thus can only be used with volatile buffers. This has meant that the “top-down” approach could not be used for studying proteins under physiological conditionsthe very conditions which are often very important for preserving a protein’s native structure and function. More complex proteins such as those with disulfide bonds present another challenge. Therefore, there is significant interest in developing novel top-down HDX methods that are applicable to all types of protein samples. In this paper, we show how top-down electron capture dissociation and subzero temperature HPLC can be combined and used for this purpose. This method keeps the back-exchange level as low as 2% and has no limitations in terms of protein type and sample solution conditions. Close to single-residue level protein structural information can be generated. The new method is validated through comparison with NMR data using calmodulin as a model protein. Its capability of determining structural changes in therapeutic antibodies (Herceptin) is also demonstrated

Standardized Protocols for Quality Control of MRM-based Plasma Proteomic Workflows

Mass spectrometry (MS)-based proteomics is rapidly emerging as a viable technology for the identification and quantitation of biological samples, such as human plasmathe most complex yet commonly employed biofluid in clinical analyses. The transition from a qualitative to quantitative science is required if proteomics is going to successfully make the transition to a clinically useful technique. MS, however, has been criticized for a lack of reproducibility and interlaboratory transferability. Currently, the MS and plasma proteomics communities lack standardized protocols and reagents to ensure that high-quality quantitative data can be accurately and precisely reproduced by laboratories across the world using different MS technologies. Toward addressing this issue, we have developed standard protocols for multiple reaction monitoring (MRM)-based assays with customized isotopically labeled internal standards for quality control of the sample preparation workflow and the MS platform in quantitative plasma proteomic analyses. The development of reference standards and their application to a single MS platform is discussed herein, along with the results from intralaboratory tests. The tests highlighted the importance of the reference standards in assessing the efficiency and reproducibility of the entire bottom-up proteomic workflow and revealed errors related to the sample preparation and performance quality and deficits of the MS and LC systems. Such evaluations are necessary if MRM-based quantitative plasma proteomics is to be used in verifying and validating putative disease biomarkers across different research laboratories and eventually in clinical laboratories

Standardized Protocols for Quality Control of MRM-based Plasma Proteomic Workflows

Mass spectrometry (MS)-based proteomics is rapidly emerging as a viable technology for the identification and quantitation of biological samples, such as human plasmathe most complex yet commonly employed biofluid in clinical analyses. The transition from a qualitative to quantitative science is required if proteomics is going to successfully make the transition to a clinically useful technique. MS, however, has been criticized for a lack of reproducibility and interlaboratory transferability. Currently, the MS and plasma proteomics communities lack standardized protocols and reagents to ensure that high-quality quantitative data can be accurately and precisely reproduced by laboratories across the world using different MS technologies. Toward addressing this issue, we have developed standard protocols for multiple reaction monitoring (MRM)-based assays with customized isotopically labeled internal standards for quality control of the sample preparation workflow and the MS platform in quantitative plasma proteomic analyses. The development of reference standards and their application to a single MS platform is discussed herein, along with the results from intralaboratory tests. The tests highlighted the importance of the reference standards in assessing the efficiency and reproducibility of the entire bottom-up proteomic workflow and revealed errors related to the sample preparation and performance quality and deficits of the MS and LC systems. Such evaluations are necessary if MRM-based quantitative plasma proteomics is to be used in verifying and validating putative disease biomarkers across different research laboratories and eventually in clinical laboratories

Analysis of Selected Sugars and Sugar Phosphates in Mouse Heart Tissue by Reductive Amination and Liquid Chromatography-Electrospray Ionization Mass Spectrometry

Sensitive and reliable analysis of sugars and sugar phosphates in tissues and cells is essential for many biological and cell engineering studies. However, the successful analysis of these endogenous compounds in biological samples by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) is often difficult because of their poor chromatographic retention properties in reversed-phase LC, the complex biological matrices, and the ionization suppression in ESI. This situation is further complicated by the existence of their multiple structural isomers in vivo. This work describes the combination of reductive amination using 3-amino-9-ethylcarbazole, with a new LC approach using a pentafluorophenyl core–shell ultrahigh performance (UP) LC column and methylphosphonic acid as an efficient tail-sweeping reagent for improved chromatographic separation. This new method was used for selected detection and accurate quantitation of the major free and phosphorylated reducing sugars in mouse heart tissue. Among the detected compounds, accurate quantitation of glyceraldehyde, ribose, glucose, glycerylaldehyde-3-phosphate, ribose-5-phosphate, glucose-6-phosphate, and mannose-6-phosphate was achieved by UPLC/multiple-reaction monitoring (MRM)-MS, with analytical accuracies ranging from 87.4% to 109.4% and CVs of ≤8.5% (<i>n</i> = 6). To demonstrate isotope-resolved metabolic profiling, we used UPLC/quadrupole time-of-flight (QTOF)-MS to analyze the isotope distribution patterns of C3 to C6 free and phosphorylated reducing sugars in heart tissues from ¹³C-labeled wild type and knockout mice. In conclusion, the preanalytical derivatization-LC/ESI-MS method has resulted in selective determination of free and phosphorylated reducing sugars without the interferences from their nonreducing structural isomers in mouse heart tissue, with analytical sensitivities in the femtomole to low picomole range

Comprehensive Analysis of Oil Sands Processed Water by Direct-Infusion Fourier-Transform Ion Cyclotron Resonance Mass Spectrometry with and without Offline UHPLC Sample Prefractionation

Oil sands processed water (OSPW) is the main byproduct of the large-scale bitumen extraction activity in the Athabasca oil sands region (Alberta, Canada). We have investigated the acid-extractable fraction (AEF) of OSPW by extraction-only (EO) direct infusion (DI) negative-ion mode electrospray ionization (ESI) on a 12T-Fourier transform ion cyclotron resonance mass spectrometer (FTICR-MS), as well as by offline ultrahigh performance liquid chromatography (UHPLC) followed by DI-FTICR-MS. A preliminary offline UHPLC separation into 8 fractions using a reversed-phase C4 column led to approximately twice as many detected peaks and identified compounds (973 peaks versus 2231 peaks, of which 856 and 1734 peaks, respectively, could be assigned to chemical formulas based on accurate mass measurements). Conversion of these masses to the Kendrick mass scale allowed the straightforward recognition of homologues. Naphthenic (C_<i>n</i>H_{2<i>n</i>+<i>z</i>}O₂) and oxy-naphthenic (C_<i>n</i>H_{2<i>n</i>+<i>z</i>}O_<i>x</i>) acids represented the largest group of molecules with assigned formulas (64%), followed by sulfur-containing compounds (23%) and nitrogen-containing compounds (8%). Pooling of corresponding fractions from two consecutive offline UHPLC runs prior to MS analysis resulted in ∼50% more assignments than a single injection, resulting in 3-fold increase of identifications compared to EO-DI-FTICR-MS using the same volume of starting material. Liquid–liquid extraction followed by offline UHPLC fractionation thus holds enormous potential for a more comprehensive profiling of OSPW, which may provide a deeper understanding of its chemical nature and environmental impact

Comparison of cytokine levels between tube types and processing protocols.

<p>Concentration of 20 cytokines in pg/ml from plasma collected in K₂EDTA tubes, P100 tubes processed with different protocols and CTAD tubes. All tubes were processed immediately after collection (T0). Brackets depict groups compared using paired t-tests and for which statistical significance * p<0.05 was found.</p

Cytokine levels at time 0.

<p>Concentration of cytokines in pg/ml from plasma collected in K₂EDTA (white bars) and P100 (black bars) tubes processed immediately after collection (T0) using protocol A. The levels of 20 cytokines detected using the Bio-Plex® Assay are shown (RANTES levels not included in the figure, concentration is off scale). Paired t-test comparisons were performed for each cytokine between tube types. * p<0.05, <b>+</b> cytokines not detected in K₂EDTA samples.</p