Accurate mass measurements using MALDI-TOF with delayed extraction.

Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry is now an essential tool in biopolymer analysis. Sensitivity and mass range are unsurpassed, but mass measurement accuracy and resolution have been limited. With delayed extraction and a reflecting analyzer, mass measurements using MALDI-TOF can be made with an accuracy of a few parts per million (ppm). It is possible to distinguish Lys from Gln in peptides, and to determine the elemental composition of smaller molecules (mass 100-500). In database searching strategies, a smaller mass window, resulting from an increase in mass accuracy, greatly decreases the number of possible candidates. Mass measurement accuracy with errors less than 5 ppm is demonstrated on a mixture of 12 peptides ranging in mass from ca. 900 to 3700 Da. Mass measurements on 13 peaks in an unseparated tryptic digest of myoglobin gave results with an overall average error less than 3.5 ppm, with a maximum error of 7 ppm.

Sensitivity and mass accuracy for proteins analyzed directly from polyacrylamide gels: implications for proteome mapping.

Matrix-assisted laser desorption ionization (MALDI) mass spectra have been obtained directly from thin-layer isoelectric focusing (IEF) gels with as little as 700 femtomoles of alpha- and beta-chain bovine hemoglobin and bovine carbonic anhydrase, and 2 picomoles of bovine trypsinogen, soybean trypsin inhibitor, and bovine serum albumin all loaded onto a single lane. By soaking the gel in a matrix solution, matrix was deposited over the entire gel surface, allowing MALDI scanning down complete lanes of the one-dimensional gel. As long as matrix crystals were deposited finely on the surface of the gel, time-lag focusing techniques were capable of ameliorating some of the mass accuracy limitations inherent in desorbing from uneven insulator surfaces with external calibration. Eleven measurements on the 5 kDa alpha-subunit proteins of lentil lectin measured over the course of 1 h and referenced to a single calibration yielded a standard deviation of 0.025%. Colloidal gold staining was found to be compatible with desorption directly from IEF and sodium dodecyl sulfate (SDS)-polyacrylamide gels. This direct approach simplifies the interface between gel electrophoresis and mass spectrometry dramatically, making the process more amenable to automation.

Dynamic modeling of electrophoretically mediated microanalysis.

A dynamic model is presented for simulation of reaction-based chemical analysis of enzymes and substrates in capillary electrophoretic systems by the methodology of electrophoretically mediated microanalysis (EMMA). The mathematical model utilizes mass balance expressions describing the time-dependent effects of electromigration, chemical reaction, and diffusional band broadening upon the concentration profiles of the various reagent and product species. The model is implemented in an iterative computer program in which the capillary is segmented into arrays of bins storing the concentration profiles of each of the chemical species. During each time increment, the effects of electrophoresis, reaction kinetics, and diffusion are calculated, and the concentrations stored in the arrays are updated. The flexibility of the model to accommodate various initial capillary conditions, sample introduction methods, and voltage programming allows diverse EMMA analyses to be simulated. The simulated results are shown to be in good qualitative agreement with experimental data for zonal injection and moving boundary EMMA determinations of leucine aminopeptidase as well as an EMMA analysis of ethanol.

C-terminal ladder sequencing via matrix-assisted laser desorption mass spectrometry coupled with carboxypeptidase Y time-dependent and concentration-dependent digestions.

The utility of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry for the analysis of C-terminal peptide ladders from carboxypeptidase Y (CPY) digestions is discussed. MALDI analysis of aliquots of an optimized time-dependent CPY digestion of ACTH 7-38 fragment allowed for the sequence of the first 19 amino acids from the C-terminus to be determined in 25 min of digestion time. A strategy for performing parallel concentration-dependent digestions on the MAL-DI plate is proven to be superior to the time-dependent approach as the method development time and practical amounts of both peptide and enzyme consumed are reduced significantly. The on-plate approach offered the same sequence information from the ACTH 7-38 fragment and was used to digest 22 peptides of various amino acid composition, size, charge, and polarity. Of the 22 peptides digested on-plate, sequence information was derived from 19 of them. A statistical analysis strategy for ladder sequencing utilizing t-statistics is offered as a method for placing confidence intervals on residue assignments.

Electrophoretically mediated microanalysis of calcium.

Electrophoretically mediated microanalysis (EMMA) was used for the determination of calcium. The faster analyte zone containing the calcium was injected spatially behind a slower zone of o-cresolphthalein complexone in a capillary electrophoresis based system. Upon application of the electric field the calcium zone was electrophoretically mixed with the reagent and product was formed. The bulk electroosmotic flow carried the product to the detector where the absorbance of the resulting complex at 575 nm was measured. Quantitation using an internal standard yielded a linear response with an R.S.D. of 8.1%. An inter-method comparison was performed with the standard bulk method and yielded results that did not significantly differ. The advantages of EMMA with respect to traditional methods were addressed.

Electrophoretically mediated microanalysis of ethanol.

Capillary electrophoresis was used to determine ethanol by the methodology of electrophoretically mediated microanalysis (EMMA). In EMMA, spatially distinct analyte and analytical reagent zones of differing electrophoretic mobility are merged under the influence of an electric field, and the resulting product is transported to the detector. The enzymatic oxidation of ethanol to acetaldehyde by alcohol dehydrogenase was utilized, and the concurrent reduction of NAD+ to NADH was monitored at 340 nm as a measure of the quantity of ethanol injected. Quantitation using an internal standard and normalization for peak migration time yielded a R.S.D. of 2.7%, and the linear range extended to that quantity of ethanol which could be reacted prior to passing by the detection window. Comparison of the EMMA technique to the Sigma spectrophotometric procedure revealed that the two methods do not yield significantly different values for the determination of ethanol. The EMMA method offered the advantages of electrophoretic mixing and miniaturization.

Mathematical treatment of electrophoretically mediated microanalysis.

A new concept in reaction-based chemical analysis is introduced and theoretically described. By utilization of the variability in electrophoretic mobilities among charged species, spatially distinct zones of chemical reagents can be electrophoretically merged under the influence of an applied electric field. Electrophoretically mediated microanalysis (EMMA) exploits this phenomenon as a basis for chemical analysis utilizing capillary electrophoretic systems. EMMA is described in terms of the four stages required for reaction-based analysis: (1) analyte and analytical reagent metering; (2) initiation of reaction; (3) control of reaction conditions and product formation; (4) detection of species whose production or depletion is indicative of the concentration or quantity of the analyte of interest. The method is illustrated by the enzymatic oxidation of ethanol to acetaldehyde by alcohol dehydrogenase with the concurrent reduction of NAD+ to NADH monitored at 340 nm. Experimental results for both substrate and enzyme determinations are shown to agree with the presented theory.

Forced expiratory volume, height, and demispan in Canadian men and women aged 55-86.

In a random cross-sectional study in London, Canada, forced expiratory volume in 1 second (FeV1.0), height, and demispan (fingerweb to central sternal notch) were measured on 182 men and 212 women of mean ages 70.4 (SD 8.5) and 71.3 (8.2). The mean FeV1.0 for men was 2.55 (0.66) l, and for women 1.80 (0.48) l and declined 29 ml.year-1 and 25 ml.year-1, respectively. The mean FeV1.0 standardized for measured height and calculated height for men was 2.54 (0.61) l and 2.55 (0.62) l in men, and 1.80 (0.46) l and 1.79 (0.45) l in women. There was no significant difference between the two methods of standardization, and the relationship of height to demispan in this independent elderly population was constant across age for both men (2.16) and women (2.17). Linear and proportional regression coefficients to predict FeV1.0 from height and demispan have been generated for this random sample aged 55 to 86 years.