Application of high-performance liquid chromatograph-electrospray ionization mass spectrometry and matrix-assisted laser-desorption ionization time-of-flight mass spectrometry in combination with selective enzymatic modifications in the characterization of glycosylation patterns in single-chain plasminogen activator

The application of high-performance liquid chromatography (HPLC), electrospray ionization mass spectrometry (ESI-MS) and matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and selective enzymatic deglycosylation treatments is demonstrated in the analysis of glycosylation patterns in recombinant Desmodus salivary plasminogen activator, a heterogeneous glycoprotein. The sample was initially digested with a proteolytic enzyme (endoproteinase Lys-C) and then further treated with either PNGase F to remove N-linked carbohydrates or a combination of neuraminidase and O-glycosidase to remove sialic acid and O-linked carbohydrates. By comparison of the LC-ESI-MS peptide maps for the fully glycosylated and deglycosylated samples, it was possible to unambiguously identify the sites of N-linked glycosylation as well a number of N-linked glycopeptides. The O-link glycopeptides, which are present at low level ( < 1%), were not detected prior to the deglycosylation, nor could changes in peptide elution in the map following deglycosylation be correlated with potential O-linked glycosylation sites.     

NIST-NPL interlaboratory pulse measurement comparison

A comparison of the pulse parameter values obtained from the pulse measurement services of the National Institute of Standards and Technology, USA, and the National Physical Laboratory, U.K., was performed. The comparison was based on the pulse parameters of amplitude, transition duration, overshoot, and undershoot (preshoot). The parameter comparison was applied to raw (measured) waveforms, corrected waveforms (if applicable), and reconstructed waveforms. The results of the comparison show that the pulse parameter values for both national laboratories are within published uncertainties.     

The effects of timing jitter in sampling systems

Timing jitter generally causes a bias (systematic error) in the amplitude estimates of sampled waveforms. Equations are developed for computing the bias in both the time and frequency domains. Two principle estimators are considered: the sample mean and the so-called Markov estimator used in some equivalent-time sampling systems. Examples are given using both real and simulated data. It is shown that the bias that results from using the sample mean as an estimator can be approximated in the frequency domain by a simple filter function. The Markov estimator is shown to asymptotically converge to the population median. It is therefore an unbiased estimator for monotonic waveforms sampled with jitter distributions having a median of zero     

Code probability distributions of A/D converters with random inputnoise

The specific architecture of an A/D converter influences the code probability distributions that result from random input noise. In particular, the output codes of successive approximation A/D converters have a spiked distribution, and its variance is half that of the corresponding input noise. In addition, the distribution has a small bias. These and other related results are derived, and are qualitatively supported by measurement data on a real 16-bit A/D converter     

A pulse measurement intercomparison

A pulse measurement intercomparison, organized by the National Institute of Standards and Technology (NIST) and conducted by the authors in their respective labs, is described. The purpose was to assess the state of the art for time-domain pulse waveform measurements in the nanosecond regime, and to find problem areas in need of better metrology support. The experiment was conducted by circulating two stable pulse generators among the five labs; participants recorded the waveforms over two different time epochs: 10.24 ns, with 10 ps sampling period and 102.4 ns with 100 ps sampling period. The data records were sent to NIST for analysis and comparison. The pulse generators that were used produce a step-like waveform with nominal high and low states of 0.5 and 0 V, respectively, transition duration of approximately 200 ps, and significant frequency components out to almost 10 GHz. The settling behavior was purposely spoiled. Some significant measurement differences were found among the five labs. The overall experiment is described, along with measurement results and conclusions     

Circulating Levels of sFlt1 Splice Variants as Predictive Markers for the Development of Preeclampsia

Angiogenic biomarkers, including soluble fms-like tyrosine kinase 1 (sFlt1), are thought to be predictors of preeclampsia onset; however, improvement is needed before a widespread diagnostic test can be utilized. Here we describe the development and use of diagnostic monoclonal antibodies specific to the two main splice variants of sFlt1, sFlt1-1 and sFlt1-14. These antibodies were selected for their sensitivity and specificity to their respective sFlt1 isoform in a capture ELISA format. Data from this pilot study suggest that sFlt1-1 may be more predictive of preeclampsia than total sFlt1. It may be possible to improve current diagnostic platforms if more specific antibodies are utilized.     

Developing linear error models for analog devices

Techniques are presented for developing linear error models for analog and mixed-signal devices. A simulation program developed to understand the modeling process is described, and results of simulations are presented. Methods for optimizing the size of empirical error models based on simulated error analyses are included. Once established, the models can be used in a comprehensive approach for optimizing the testing of the subject devices. Models are developed using data from a group of 13-bit A/D converters and compared with the simulation results     

Bounds on frequency response estimates derived from uncertain stepresponse data

The frequency response of a system can be estimated from measurements of its step response; however, many error sources affect the accuracy of such estimates. This paper investigates the effects of uncertainty in the knowledge of the step response. Methods for establishing uncertainty bounds for the frequency response estimates are developed, based on the corresponding time-domain uncertainties associated with the measured step response. Two methods are described. One method produces bounds that are often very conservative. The other method produces bounds that are more realistic. End effects that influence the bounds are also considered. A simulation example and an application of the bounds are presented     

Cutting the high cost of testing

A modeling approach to the overly long testing of analog and mixed-signal devices that saves substantially on time and cost is described. The discussion focuses on the particular case of a 13 bit analog-to-digital converter (ADC). The problems that arise in testing ADCs are identified, showing that the success of the test method depends critically on the quality of the model. Two types of models are examined, physical-sensitivity-based models and empirical-learning-based models, and it is noted that the latter are especially attractive for performance-testing applications like the ADC example. An 18-parameter model of the 13 bit ADC was developed using a combination of physical and empirical modeling techniques and was highly successful. With an array process to speed up the computations, the computational overhead can be kept below 1 s per device, so the test time, which is reduced by a factor of 128, becomes negligible     

A fast-pulse oscilloscope calibration system

A system is described for calibrating high-bandwidth oscilloscopes using pulse signals. The fast-pulse oscilloscope calibration system (FPOCS) is to be used to determine the step response parameters for digitizing oscilloscopes having bandwidths of ~20 GHz. The system can provide measurement traceability to standards maintained at the U.S. National Institute of Standards and Technology (NIST). It comprises fast electrical step generation hardware, a personal computer (PC) and software, and a reference waveform, i.e., a data file containing an estimate of the step generator output signal. The reference waveform is produced by prior measurement by NIST of the step generator output signal (calibration step signal). When the FPOCS is in use, the calibration step signal is applied to the device under test, which is an oscilloscope sampling channel. The measured step waveform is corrected for timebase errors, then the reference waveform is deconvolved from it. The results are impulse, step, and frequency response estimates, and their associated parameters (e.g., transition duration, transition amplitude, -3 dB bandwidth) and uncertainties. The system and its components are described, and preliminary test results are presented