Very high pressure gradient LC/MS/MS.

A very high pressure liquid chromatography (VHPLC) system was constructed by modifying a commercially available pump in order to achieve pressures in excess of 1,200 bar (17,500 psi). A computer-controlled low-pressure mixer was used to generate solvent gradients. Protein digests were rapidly analyzed by reversed-phase VHPLC with linear solvent gradients coupled to either a tandem mass spectrometer using electrospray ionization or a UV/visible detector. The separations were performed at pressures ranging from 790 (11,500 psi) to 930 bar (13,500 psi) in 22-cm-long capillary columns packed with C18-modified 1.5-microm nonporous silica particles. A digest of bovine serum albumin (BSA) was analyzed by the VHPLC system connected to a mass spectrometer in MS mode. An analysis of 12.5 fmol of sample gave signal-to-noise ratios of tryptic peaks greater than 10:1 in the base peak plot mass chromatogram. This system was also used to analyze a proteolytic digest of a rat liver protein excised from a 2-D gel separation of a liver tissue lysate. For this analysis, the mass spectrometer was set up to perform data-dependent scanning (automated switching from MS mode to MS/MS mode when a peak was detected) for peptide sequencing and protein identification by database searching. The results of this analysis are compared to an analysis performed on the same sample using the nanoelectrospray-MS/MS technique. Though both techniques were able to identify the unknown protein, the VHPLC method gave twice as many sequenced peptides as nanoelectrospray and improved the signal-to-noise ratio of the spectra by at least a factor of 10. Direct comparisons with nanoelectrospray for MS and MS/MS data acquisition from a BSA digest were made. These comparisons show enhancements of greater than 20-fold for VHPLC over nanoelectrospray. In addition, the VHPLC/MS/MS data acquisition was accomplished in an automated manner.

A hybrid of exponential and gaussian functions as a simple model of asymmetric chromatographic peaks.

A hybrid of exponential and Gaussian functions is developed as a model of asymmetric peak profiles. This exponential-Gaussian hybrid function (EGH) is mathematically simple, numerically stable, and its parameters are readily determined by making graphical measurements and applying simple equations. Furthermore, the statistical moments of the EGH function can be accurately approximated (within+/-0.15% error) at any level of asymmetry using formulae that are easily programmed into a computer. These features of the EGH make it very easy to implement by most chromatographers. The EGH serves as a useful alternative to the exponentially modified Gaussian (EMG) for modeling slightly asymmetric peaks since the two models produce nearly the same profile at relatively low asymmetries. The EGH also serves as an addition to the extensive list of alternative models that are sometimes better than the EMG at describing highly asymmetric peaks. A comparison between EMG and EGH curves at various asymmetries is made by analysis of toluene, phenylalanine, and pyridine on a reversed-phase liquid chromatographic system.

Spatial and temporal progressions of spatial statistical moments in linear chromatography.

Generalizations of existing models of chromatography allow the spatial and temporal progressions of all spatial statistical moments in linear chromatography to be given as the solution to a set of ordinary differential equations. Basic strategies of simplifying these equations are described.

High resolution of oligosaccharide mixtures by ultrahigh voltage micellar electrokinetic capillary chromatography.

Oligosaccharide mixtures released from ribonuclease B and human IgG have been separated using micellar electrokinetic capillary chromatography operated at 100 kV. The resolution of these closely related analytes at this high voltage was found to be superior to that obtained at 20 kV, a voltage which is ordinarily used in most capillary electrophoresis separations.

Theoretical investigation of the spatial progression of temporal statistical moments in linear chromatography.

Migration and dispersion in chromatography are modeled by analogy to an effective eddy diffusion process. On the basis of this model, the spatial rates of temporal statistical moment change are derived for general chromatography in linear media. In most practical cases, these equations can be simplified so that temporal statistical moments can be calculated by solving a system of ordinary differential equations that depend only on the local HETP, solute velocity, and initial values of the temporal statistical moments. The calculations of temporal centroid, temporal variance, temporal skew, and temporal excess are demonstrated for the case of linear solvent strength gradients. It is shown for the case of temporally invariant separation environments, such as isocratic liquid chromatographic systems and isothermal gas chromatographic systems, that temporal variance contributions are spatially additive and that the temporal third normalized central moment is unaffected by spatial variations in the medium. A refined explanation is given for how peak symmetry is improved in gradient forms of chromatography.

Ultrahigh-pressure reversed-phase capillary liquid chromatography: isocratic and gradient elution using columns packed with 1.0-micron particles.

Fused-silica capillaries with inner diameters of 33 microns and lengths of 25-50 cm are slurry-packed with 1.0-micron nonporous octadecylsilane-modified (C18) silica spheres. These columns are used to perform ultrahigh-pressure reversed-phase liquid chromatographic analyses in both isocratic and gradient elution modes. Mobile-phase pressures as high as 5000 bar (72,000 psi) are applied to column inlets to generate more than 200,000 theoretical plates in 6 min (k' approximately 1) for small, organic analytes. Average capacity factors of analytes are found to increase linearly with applied pressure. An electrically driven constant-flow syringe pump capable of generating mobile-phase pressures as high as 9000 bar (130,000 psi) is described. This pump is used in conjunction with an exponential dilution method for the gradient separation of peptides from a tryptic digest on a 27-cm-long capillary packed with 1.0-micron particles. A peak capacity of 300 is demonstrated for a 30-min analysis.

Automated measurement of peak widths for the determination of peak capacity in complex chromatograms.

The peak capacity was measured for an ultrahigh-pressure gradient elution chromatogram of a fluorescently tagged tryptic digest of ovalbumin. The peak widths in the chromatogram were determined by measuring the peak height and the second derivative at the peak maximum. This approach for measuring peak widths was programmed into a computer, and the software accurately determined the general progression of peak widths by measuring 47 peaks throughout the chromatogram in under 10 s. Peak capacity was determined by taking the definite integral of the plot of reciprocal base peak width versus retention time. This calculation of peak capacity is a linear transformation with respect to separation space, so the method is more rigorously accurate than previous methods. The peak capacity for the chromatogram was calculated to be 316.

Ultrahigh-voltage capillary zone electrophoresis.

An ultrahigh-voltage capillary electrophoresis system was built to demonstrate the possibility of extending the applied potential and thus the separation power of capillary electrophoresis. A commercial 30-kV power supply was extensively modified in order to provide electrical potentials up to 120 kV. A unique electrical shielding system was developed to prevent capillary breakdown and corona or spark discharges. Electrophoretic studies using a mixture of peptide standards, as well as a complex mixture of peptides obtained from a protein digest, showed that the numbers of theoretical plates achieved increase linearly with applied voltage. Theoretical plate counts ranging from 2.7 to 6.1 million plates were obtained for peptides in a separation done at 120 kV. Resolution also increased with the square root of applied voltage, as predicted by theory.

Pressure-induced retention variations in reversed-phase alternate-pumping recycle chromatography.

The progressions of peak width and peak separation in reversed-phase alternate-pumping (AP) recycle chromatography are found to be inconsistent with conventional chromatographic theory. These discrepancies are explained by subtle pressure-induced variations of solute retention that become amplified by AP recycling. The presence of these retention variations is demonstrated by multiply injecting a single solute into an AP system at offset times. As the serially injected peaks are recycled, the separation time between the peaks is shown to vary significantly, indicating that the retention of the solute is dependent upon the position of the peak. A new model of chromatographic retention that appropriately accounts for this variable retention is presented. When this retention model is applied to an AP system for the binary separation of phenylalanine and a pentadeuterated phenylalanine, the model accurately describes the experimentally observed progressions of peak width and peak separation. Furthermore, the retention model predicts that the improvement of resolution in AP recycling closely matches the expectations of conventional theory, so the effectiveness of AP recycling is not significantly compromised by the variations in retention.

Comprehensive two-dimensional high-performance liquid chromatography for the isolation of overexpressed proteins and proteome mapping.

A two-dimensional liquid chromatographic system is described here which uses size-exclusion liquid chromatography (SEC) followed by reversed-phase liquid chromatography (RPLC) to separate the mixture of proteins resulting from the lysis of Escherichia coli cells and to isolate the proteins that they produce. The size-exclusion chromatography can be conducted under either denaturing or nondenaturing conditions. Peaks eluting from the first dimension are automatically subjected to reversed-phase chromatography to separate similarly sized proteins on the basis of their various hydrophobicities. The RPLC also serves to desalt the analytes so that they can be detected in the deep ultraviolet region at 215 nm regardless of the SEC mobile phase used. The two-dimensional (2D) chromatograms produced in this manner then strongly resemble the format of stained 2D gels, in that spots are displayed on a X-Y axis and intensity represents quantity of analyte. Following chromatographic separation, the analytes are deposited into six 96-well (576 total) polypropylene microtiter plates via a fraction collector. Interesting fractions are analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS) or electrospray mass spectrometry (ESI/MS) depending on sample concentration, which both yield accurate (2 to 0.02%) molecular weight information on intact proteins without any additional sample preparation, electroblotting, destaining, etc. The remaining 97% of a fraction can then be used for other analyses, such Edman sequencing, amino acid analysis, or proteolytic digestion and sequencing by tandem mass spectrometry. This 2D HPLC protein purification and identification system was used to isolate the src homology (SH2) domain of the nonreceptor tyrosine kinase pp60c-src and beta-lactamase, both inserted into E. coli, as well as a number of native proteins comprising a small portion of the E. coli proteome.

Separation and identification of peptides in single neurons by microcolumn liquid chromatography-matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and postsource decay analysis.

Microcolumn liquid chromatography (LC) was interfaced with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for separation and identification of peptides present in single neurons from the brain of the snail Lymnaea stagnalis. The nanoliter microcolumn LC effluent, mixed off-line with nanoliter matrix solution, was deposited onto the sample target every 60 s, producing fractions of approximately 145 nL in volume, which, upon drying, produced spots of approximately 1 mm in size. At the end of the chromatographic separation, fractions from the sample target were scanned by MALDI-TOF-MS. Identification of peptide peaks was achieved on the basis of LC elution order and mass information. Further identification based on sequence information was carried out for a native peptide fractionated by microcolumn LC from a single neuron with the postsource decay technique.

Quantal corelease of histamine and 5-hydroxytryptamine from mast cells and the effects of prior incubation.

Corelease of histamine and 5-hydroxytryptamine from individual mast cells has been measured with fast-scan cyclic voltammetry using a carbon-fiber electrode placed next to a single cell. Release events, induced by exposure of the cells to the calcium ionophore, A23187, were resolved at the level of individual exocytotic events. Changes in the relative concentrations secreted from individual granules were observed after incubation with 5-hydroxytryptamine, histamine, and tryptophan. In contrast, an alteration in individual cell content after such incubations, analyzed with capillary chromatography, was only found after incubation with 5-hydroxytryptamine. Cells incubated with 5-hydroxytryptamine or its precursor, tryptophan, released more 5-hydroxytryptamine and less histamine per secretory event relative to controls. Coincubation of the cells with pargyline and 5-hydroxytryptamine further reduced the release of histamine. Since cell content of histamine is unchanged, the reduction in its release must be due to its displacement to a nonreleasable compartment induced by 5-hydroxytryptamine granular uptake. Incubation with histamine increased histamine secretion and, surprisingly, also increased 5-hydroxytryptamine release without changing its cell content. This result is consistent with a relaxation of the storage matrix accompanying histamine granular uptake allowing more 5-hydroxytryptamine to be released. These results demonstrate that the intragranular mode of storage as well as granular uptake of biogenic amines affects the stoichiometry of their release.

Lowering the UV Absorbance Detection Limit in Capillary Zone Electrophoresis Using a Single Linear Photodiode Array Detector.

A new approach for lowering the UV absorbance detection limit in capillary electrophoresis is presented. This approach involves the use of a photodiode array in which each of the diodes in the array is treated as an independent detector. Over the course of a run, therefore, an electropherogram is generated for each diode in the array. Averaging the electropherograms generated from 1500 diodes in a diode array resulted in a signal-to-noise ratio 85 times that of an electropherogram generated from any one diode in the array. These signal-to-noise improvements are discussed, and the detection limits are compared to the detection limits obtained from a commercial single-point detector. The array detector improves the detection limit by a factor of 3.8 (±0.4).

Determination of enzyme activity in single bovine adrenal medullary cells by separation of isotopically labeled catecholamines.

A microcolumn liquid chromatography method for determining norepinephrine (NE), epinephrine (E), and phenylethanolamine N-methyltransferase (PNMT) enzyme activity in single bovine adrenal medullary cells is presented. Single cells were isolated and treated with excess deuterated substrate, D3-NE (0.05 mM) for enzyme reaction. After 6 h, the reaction was quenched and the product, D3-E, was quantified along with endogenous NE and E. Separation and detection of deuterated and protiated NE and E were achieved with microcolumns (110-125 cm long, 25 microns inner diameter) packed with 3 microns octadecylsilane-modified particles and operated with amperometric detection. Of the 33 cells reported, most cells containing predominantly E have enzyme activity while cells containing predominantly NE and cells containing a mixture of both NE and E show no enzyme activity. After incubation with 10 microM hydrocortisone, of the 17 cells reported, most cells containing predominantly E and cells containing a mixture of both NE and E have enzyme activity while cells containing predominantly NE have no enzyme activity. Detection limits for NE and E were 42 and 48 amol, respectively.

Sensitive analysis of [D-Pen2,5]enkephalin in rat serum by capillary electrophoresis and laser-induced fluorescence detection.

A highly sensitive analytical method based on capillary zone electrophoresis (CZE) coupled with a laser-induced fluorescence (LIF) detector was explored for the analysis of [D-Pen2,5]enkephalin (DPDPE) in rat serum. DPDPE and the internal standard Phe-Leu-Glu-Glu-Ile (P9396) were extracted from serum samples with C18 solid-phase extraction disk cartridges, followed by derivatization with tetramethylrhodamine-5-isothiocyanate (TRITC) isomer G before introduction onto the capillary column. Complete resolution of DPDPE and the internal standard from other serum components was achieved within 20 min on a 140 cm x 50 microns I.D. capillary column with borate buffer (25 mM. pH 8.3). With the current method, it is possible to detect 1.3E-18 mol of DPDPE on column. The results suggest that CZE-LIF is a promising method for the sensitive and specific quantitation of therapeutic peptides in biological matrices.

Two-dimensional SEC/RPLC coupled to mass spectrometry for the analysis of peptides.

A two-dimensional liquid chromatography system is described here which uses size exclusion liquid chromatography (SEC) followed by reversed phase liquid chromatography (RPLC) to separate the mixture of peptides resulting from the enzymatic digestion of a protein. A novel LC/LC interface, using two RPLC columns in parallel rather than storage loops, joins the two chromatographic dimensions. This new interface design permits the use of conventional analytical diameter HPLC columns, 7.8 mm for SEC and 4.6 mm for RPLC, making construction and maintenance of this system very easy. The reversed phase chromatography utilizes 1.5 microns diameter, nonporous C-18 modified silica particles, which produce fast and efficient analyses. Following the high-resolution two-dimensional chromatographic separation, an electrospray mass spectrometer detects the peptide fragments. The mass spectrometer scans a 2000 m/z range to identify the analytes from their molecular weights. The analyses of tryptic digests of ovalbumin and serum albumin are each described.

A multidimensional approach to protein characterization.

When mass spectrometry (MS) is used to study protein primary structure, it is used in a "static" mode. That is, the information is derived from a single MS or MS-MS spectrum. Information about more complex protein structure or protein interactions can also be gained via MS. If a series of mass spectra is collected as something else in the experiment is changing, we increase the "dimensionality" of the MS data. For example, measuring mass spectra as a function of time after exposure of a protein to deuterated solvents can provide information about protein structure. Likewise, by measuring mass spectra of a protein as the concentration of a binding ligand is changed, one can infer the stoichiometry of the complex. Another important, but fundamentally different way of increasing the dimensionality of mass spectral data is by coupling the mass spectrometer to a one- or two-dimensional separation technique.

Comprehensive on-line LC/LC/MS of proteins.

This is a description of a comprehensive two-dimensional liquid chromatography (LC) system for the separation of protein mixtures. This system uses cation-exchange chromatography followed by reversed-phase chromatography (RPLC). The two LC systems are coupled by an eight-port valve equipped with two storage loops and under computer control. The RPLC effluent is sampled by both a UV detector and an electrospray mass spectrometer. In this way, complex mixtures of large biomolecules can be rapidly separated, desalted, and analyzed for molecular weight in less than 2 h. The system's utility is demonstrated with a mixture of standards and an Escherichia coli cell lysate.

Ultrahigh-pressure reversed-phase liquid chromatography in packed capillary columns.

The use of extremely high pressures in liquid chromatography can improve the efficiency and reduce analysis time for columns packed with small particles. In this work, fused-silica capillaries with inner diameters of 30 microns are slurry packed with 1.5 microns nonporous octadecylsilane-modified silica particles. These columns are prepared in lengths up to 66 cm with packing pressures as high as 4100 bar (60,000 psi). Near the optimum flow rate, columns generate as many as 300,000 theoretical plates for lightly retained compounds (k' < 0.5) and over 200,000 plates for more retained compounds (k' approximately 2). These translate to plate heights (Hmin) as low as 2.1 microns. The pressures required to run at optimum flow rates are on the order of 1400 bar (20,000 psi). Analysis times at these pressures are on the order of 30 min (k' approximately 2) and can be reduced to less than 10 min at higher than optimum flow rates. Capacity factors are observed to increase linearly with applied pressure.

Rapid separation and characterization of protein and peptide mixtures using 1.5 microns diameter non-porous silica in packed capillary liquid chromatography/mass spectrometry.

Octadecyl-modified 1.5 microns diameter non-porous silica particles were packed in 150 microns i.d. (360 microns o.d.) capillaries with lengths of 20 cm which were used to separate proteins and peptides generated from enzymatic digests of proteins. Gradients were produced using an exponential dilution method at pressures of 520 Bar (7500 psi) and electrospray ionization mass spectrometry was used for detection. This system was similar to packed capillary perfusion chromatography with respect to chromatographic resolution and analysis time and had a limit of detection comparable to traditional packed capillaries which use 5 microns diameter porous particles. The analyses required as little as 250 femtomol of protein or 500 femtomol of peptide on-column in approximately 30 min. This technique was then applied to verify the existence of an overexpressed protein in an E. coli cell lysate and to confirm the presence of four glycoforms of a peptide generated in the proteolytic digest of an antibody.