Top-Down and Bottom-Up Identification of Proteins by Liquid Extraction Surface Analysis Mass Spectrometry of Healthy and Diseased Human Liver Tissue

Liquid extraction surface analysis mass spectrometry (LESA MS) has the potential to become a useful tool in the spatially-resolved profiling of proteins in substrates. Here, the approach has been applied to the analysis of thin tissue sections from human liver. The aim was to determine whether LESA MS was a suitable approach for the detection of protein biomarkers of nonalcoholic liver disease (nonalcoholic steatohepatitis, NASH), with a view to the eventual development of LESA MS for imaging NASH pathology. Two approaches were considered. In the first, endogenous proteins were extracted from liver tissue sections by LESA, subjected to automated trypsin digestion, and the resulting peptide mixture was analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) (bottom-up approach). In the second (top-down approach), endogenous proteins were extracted by LESA, and analyzed intact. Selected protein ions were subjected to collision-induced dissociation (CID) and/or electron transfer dissociation (ETD) mass spectrometry. The bottom-up approach resulted in the identification of over 500 proteins; however identification of key protein biomarkers, liver fatty acid binding protein (FABP1), and its variant (Thr→Ala, position 94), was unreliable and irreproducible. Top-down LESA MS analysis of healthy and diseased liver tissue revealed peaks corresponding to multiple (~15–25) proteins. MS/MS of four of these proteins identified them as FABP1, its variant, α-hemoglobin, and 10 kDa heat shock protein. The reliable identification of FABP1 and its variant by top-down LESA MS suggests that the approach may be suitable for imaging NASH pathology in sections from liver biopsies. Graphical Abstract

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Fast,simple, and sensitive high‐performance liquid chromatography method for measuring vitamins A and E in human blood plasma

Vitamins A and E are fat‐soluble vitamins that play important roles in several physiological processes. Monitoring their concentrations is needed to detect deficiency and guide therapy. In this study, we developed a high‐performance liquid chromatography method to measure the major forms of vitamin A (retinol) and vitamin E (α‐tocopherol and γ‐tocopherol) in human blood plasma. Vitamins A and E were extracted with hexane and separated on a reversed‐phase column using methanol as the mobile phase. Retinol was detected by ultraviolet absorption, whereas tocopherols were detected by fluorescence emission. The chromatographic cycle time was 4.0 min per sample. The analytical measurement range was 0.03–5.14, 0.32–36.02, and 0.10–9.99 mg/L for retinol, α‐tocopherol, and γ‐tocopherol, respectively. Intr‐aassay and total coefficient of variation were <6.0% for all compounds. This method was traceable to standard reference materials offered by the National Institute of Standards and Technology. Reference intervals were established using plasma samples collected from 51 healthy adult donors and were found to be 0.30–1.20, 6.0–23.0, and 0.3–3.2 mg/L for retinol, α‐tocopherol, and γ‐tocopherol, respectively. In conclusion, we developed and validated a fast, simple, and sensitive high‐performance liquid chromatography method for measuring the major forms of vitamins A and E in human plasma.     

Fourier series and the Lubkin W-transform

We discuss the effect of a particular sequence acceleration method, the Lubkin W-transform, on the partial sums of Fourier series. We consider a very general class of functions with a single jump discontinuity, and prove that this method fails on a large set of points. The authors were partially supported by the Kansas State University REU and NSF grant GOMT530725.     

Taxonomy of singers used as subjects in scientific research

Criteria for determining the classification of singers in research has posed numerous problems for both researchers and their readership. Previous attempts at creating usable categories have failed because of disagreement within the professions involved in such research. The object of this paper is to explore and suggest a system in which scientific research related to singing is based on a quantifiable singing standard. Use of the systematic categorization set out in this paper will help to alleviate the questions regarding performance abilities of singers used as subjects in scientific projects and lead to more credible results. Nine singer categories have been consolidated and based on proven performance achievement. These categories range from superstar (household name) to local community and amateur singers with comprehensive subcategories under each topic for each type of singer, that is, opera, pop, jazz, etc.     

Elemental imaging of MRI contrast agents: benchmarking of LA-ICP-MS to MRI

Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) has been used to map the spatial distribution of magnetic resonance imaging (MRI) contrast agents (Gd-based) in histological sections in order to explore synergies with in vivo MRI. Images from respective techniques are presented for two separate studies namely (1) convection enhanced delivery of a Gd nanocomplex (developmental therapeutic) into rat brain and (2) convection enhanced delivery, with co-infusion of Magnevist (commercial Gd contrast agent) and Carboplatin (chemotherapy drug), into pig brain. The LA technique was shown to be a powerful compliment to MRI not only in offering improved sensitivity, spatial resolution and signal quantitation but also in giving added value regarding the fate of administered agents (Gd and Pt agents). Furthermore simultaneous measurement of Fe enabled assignment of an anomalous contrast enhancement region in rat brain to haemorrhage at the infusion site.     

Fractional conductance quantization in metallic nanoconstrictions under electrochemical potential control

We study the electrical conductance of gold nanoconstrictions by controlling the electrochemical potential. At positive potentials, the conductance is quantized near integer multiples of G0(2e(2)/h) as shown by well-defined peaks in the conductance histogram. Below a certain potential, however, additional peaks near 0.5G(0) and 1. 5G(0) appear in the histogram. The fractional conductance steps are as stable and well defined as the integer steps. The experimental data are discussed in terms of electrochemical-potential-induced defect scattering and Fermi energy shift, but a complete theory of the phenomenon is yet to be developed.     

Dried Blood Spot Proteomics: Surface Extraction of Endogenous Proteins Coupled with Automated Sample Preparation and Mass Spectrometry Analysis

Dried blood spots offer many advantages as a sample format including ease and safety of transport and handling. To date, the majority of mass spectrometry analyses of dried blood spots have focused on small molecules or hemoglobin. However, dried blood spots are a potentially rich source of protein biomarkers, an area that has been overlooked. To address this issue, we have applied an untargeted bottom-up proteomics approach to the analysis of dried blood spots. We present an automated and integrated method for extraction of endogenous proteins from the surface of dried blood spots and sample preparation via trypsin digestion by use of the Advion Biosciences Triversa Nanomate robotic platform. Liquid chromatography tandem mass spectrometry of the resulting digests enabled identification of 120 proteins from a single dried blood spot. The proteins identified cross a concentration range of four orders of magnitude. The method is evaluated and the results discussed in terms of the proteins identified and their potential use as biomarkers in screening programs.

Top-Down LESA Mass Spectrometry Protein Analysis of Gram-Positive and Gram-Negative Bacteria

We have previously shown that liquid extraction surface analysis (LESA) mass spectrometry (MS) is a technique suitable for the top-down analysis of proteins directly from intact colonies of the Gram-negative bacterium Escherichia coli K-12. Here we extend the application of LESA MS to Gram-negative Pseudomonas aeruginosa PS1054 and Gram-positive Staphylococcus aureus MSSA476, as well as two strains of E. coli (K-12 and BL21 mCherry) and an unknown species of Staphylococcus. Moreover, we demonstrate the discrimination between three species of Gram-positive Streptococcus (Streptococcus pneumoniae D39, and the viridans group Streptococcus oralis ATCC 35037 and Streptococcus gordonii ATCC35105), a recognized challenge for matrix-assisted laser desorption ionization time-of-flight MS. A range of the proteins detected were selected for top-down LESA MS/MS. Thirty-nine proteins were identified by top-down LESA MS/MS, including 16 proteins that have not previously been observed by any other technique. The potential of LESA MS for classification and characterization of novel species is illustrated by the de novo sequencing of a new protein from the unknown species of Staphylococcus.

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The boundary-residual method for three-dimensional homogeneous field problems with boundaries of arbitrary geometry

Boundary-residual methods are extended to include three-dimensional homogeneous field problems applicable to millimeter-wave problems. Their connection to pointmatching methods is shown, and the reason why pointmatching fails and boundary residual methods do not is explained. A numerical example is used to demonstrate the validity of these methods. A discussion of the limitations of these methods is also included.     

A computational method for the indefinite quadratic programming problem

We present an algorithm for the quadratic programming problem of determining a local minimum of ?(x)=$\text{1}\text{2}$x^TQx+c^Tx such that A^Tx?b where Q ymmetric matrix which may not be positive definite. Our method combines the active constraint strategy of Murray with the Bunch-Kaufman algorithm for the stable decomposition of a symmetric matrix. Under the active constraint strategy one solves a sequence of equality constrained problems, the equality constraints being chosen from the inequality constraints defining the original problem. The sequence is chosen so that ?(x) continues to decrease and x remains feasible. Each equality constrained subproblem requires the solution of a linear system with the projected Hessian matrix, which is symmetric but not necessarily positive definite. The Bunch-Kaufman algorithm computes a decomposition which facilitates the stable determination of the solution to the linear system. The heart of this paper is a set of algorithms for updating the decomposition as the method progresses through the sequence of equality constrained problems. The algorithm has been implemented in a FORTRAN program, and a numerical example is given.