Edward Voigtman

Mapping protein energy landscapes with amide hydrogen exchange and mass spectrometry: I. A generalized model for a two-state protein and comparison with experiment

Protein amide hydrogen exchange (HDX) is a convoluted process, whose kinetics is determined by both dynamics of the protein and the intrinsic exchange rate of labile hydrogen atoms fully exposed to solvent. Both processes are influenced by a variety of intrinsic and extrinsic factors. A mathematical formalism initially developed to rationalize exchange kinetics of individual amide hydrogen atoms is now often used to interpret global exchange kinetics (e.g., as measured in HDX MS experiments). One particularly important advantage of HDX MS is direct visualization of various protein states by observing distinct protein ion populations with different levels of isotope labeling under conditions favoring correlated exchange (the so‐called EX1 exchange mechanism). However, mildly denaturing conditions often lead to a situation where the overall HDX kinetics cannot be clearly classified as either EX1 or EX2. The goal of this work is to develop a framework for a generalized exchange model that takes into account multiple processes leading to amide hydrogen exchange, and does not require that the exchange proceed strictly via EX1 or EX2 kinetics. To achieve this goal, we use a probabilistic approach that assigns a transition probability and a residual protection to each equilibrium state of the protein. When applied to a small protein chymotrypsin inhibitor 2, the algorithm allows complex HDX patterns observed experimentally to be modeled with remarkably good fidelity. On the basis of the model we are now in a position to begin to extract quantitative dynamic information from convoluted exchange kinetics.

Statistical properties of limit of detection test statistics.

There are a number of possible ways to define the instrumental limit of detection (LOD) figure of merit and in the present work we define four such sample test statistics and derive their probability density functions (PDFs). Although the derived PDFs were found to be irreducible integrals, they are easily evaluated via numerical integration and can be used to obtain expectation values, population precisions and confidence intervals. Monte Carlo simulation methods were used to prepare normalized histograms of one million LOD variates each, for homoscedastic linear calibration curve systems, and these were found to be in excellent agreement with the numerically obtained PDFs and associated statistics. The software used in all aspects of the present work is available for free, with full, commented source code, and is designed to facilitate exploration of calibration curve systems of immediate interest to the reader.

Gated Peak Integration versus Peak Detection in White Noise

The optimum gated integration windows for a variety of common peak shapes in stationary, white noise are derived by means of simple linear systems theory. Optimum windows are found to provide signal-to-noise ratios less than 10% lower than the matched filter results. The confusion between peak detection and narrow gate window integration is discussed, and it is demonstrated that both are inferior to optimum gated integration. It is also shown that the matched filter result is the appropriate benchmark against which proposed new detection techniques should be compared.

COMPUTER-SIMULATIONS IN SPECTROMETRY

Abstract A commercially available simulation computer program has been augmented by the addition of libraries of Jones and Mueller calculus component blocks, enabling the program to handle mixed systems consisting of both electronic signal processing blocks and optical component blocks. The program and libraries constitute a “scientific block diagram program”. The scope and power of the synthesis are demonstrated by application to both graphite tube furnace electrothermal atomization atomic absorption spectrometry and laser-excited liquid chromatographic detection of optical activity.

Mueller Calculus Spectrometric Simulations of Thermal Lensing

A commercially available microcomputer-based simulation program, augmented with more than 130 custom-programmed component blocks, has been shown to allow the seamless integration of both optical and signal processing components on a computer screen worksheet. The libraries of custom component blocks allow scientific block diagrams, representing a variety of spectrometric techniques, to be “brought to life” as functional technique simulations. The libraries of blocks are applied to the qualitative and quantitative modeling of several published thermal lensing instruments, with the achievement of excellent results.

Stray light effects in Zeeman atomic absorption spectrometry

Abstract A transverse Zeeman atomic absorption spectrometer has been modeled using a polychromatic, optical calculus simulation software program. The models were found to be realistic and greatly facilitated the study of the effects of various Zeeman spectrometer parameters and their interactions. The behavior of the Zeeman spectrometer calibration curves, in the presence of three different types of stray light, was modeled. From the initial results, it seems most likely that the major stray light source in real Zeeman experiments is due to polychromaticity of the light source, even when the source profile is relatively narrow in spectral bandwidth.

Gated Integration of Transient Signals in 1/ f Noise

The Mueller optical calculus is used to model common spectrometric experimental schemes. The effects of source and preamplifier noise on the precision of the measurement are investigated, and the effect of gate width variation for gated integration of Gaussian signal peaks in the presence of white or 1/f type noise is evaluated. The results indicate that, for integration of a transient absorbance signal, the choice of gate width depends upon the type of noise, but not its source. For a transmittance (or emission) type of experiment, both the type and the source of the noise determine the gate width to be used for integration of the signal.

Multivariate curve resolution in liquid chromatography - resolving two-way multi-component data using a Varimax extended rotation

The Varimax extended rotations (VER) have been proposed as a new method to mathematically resolve severely overlapped peaks in chromatographic experiments that produce bilinear data. VER employs a four-step procedure to determine the relative concentration and identity of the components that comprise a severely overlapped chromatographic peak. In the first step, the data are pre-processed to ensure that they are in a form suitable for multivariate curve resolution. The second step involves principal component analysis, which reduces the dimensionality of the data matrix while simultaneously retaining the information present in the data. In the third step, a new coordinate system is developed for the data using a Varimax rotation followed by a so-called extended rotation, which assists in identifying the so-called pure regions in the peak. Identifying these regions is crucial to rotating the concentration and spectral matrices towards a solution. The fourth step utilizes alternating least squares (ALS) to improve the estimates of the concentration and spectral profiles of each component. Results from real and simulated data are used to illustrate the efficacy and simplicity of the proposed method.

Signal processing of transient atomic absorption signals

Abstract This article is an electronic publication in Spectrochimica Acta Electronica (SAE), the electronic section of Spectrochimica Acta Part B (SAB). The hard copy text is accompanied by a disk with a demonstration version of the simulation program, libraries of electronic and optical component blocks, simulation models, manual, and other files. Absorbance signals for electrothermal atomization atomic absorption spectroscopy (ETA-AAS) were generated digitally and the effect of various types and sources of noise upon the precision of the absorbance measurement was evaluated by numerical calculation. Peak area measurement, peak height measurement, and matched filtering were used for processing these signals. The performance of these three techniques in the presence of various types of noises and the sensitivity of each to small variation in the atomization conditions was calculated. It is demonstrated that significant improvement in signal-to-noise ratios can be realized by application of appropriate signal processing methods. The results also indicate that one of the principal causes for loss of precision could be the variation in the heating characteristics of the furnace.

Temporal and Spectral Characteristics of the Output of an Excimer Laser

The output of a Questek Model 2210 excimer laser, operated with XeCl fill, is characterized in both the time and frequency domains. Power spectra are obtained through use of the fast Fourier transform which show that the output is essentially white in character. Furthermore, noise power diminishes with repetition rate, reaching a broad minimum around 40 Hz. Analysis in the time domain shows a “roughly Gaussian” distribution. Movement of optically opaque (“thick”) particles in the path of the laser beam is shown to increase the variance of the noise, but not its low-frequency content.