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D Figeys

Publications and source records attributed to D Figeys.

33 records · Page 2Linked to original sources
PubMed1998

High sensitivity analysis of proteins and peptides by capillary electrophoresis-tandem mass spectrometry: recent developments in technology and applications.

Analytical biochemistry, in particular the analysis of regulatory proteins that control biological systems and pathways, is dependent on methods of ever-increasing sensitivity. Capillary electrophoresis (CE) has long been recognized as an ultrasensitive analytical technique. In spite of the high sensitivity, CE has not penetrated protein discovery research as a standard analytical method. In this review article we summarize recent technical developments which have significantly enhanced CE as a tool for the analysis of trace amounts of proteins. Specifically, we review recent advances in the development and application of capillary electrophoresis-mass spectrometry (CE-MS) and on-line analyte concentration techniques, and introduce the emerging field of microfluidics as a front end to mass spectrometry (MS).

Electrophoresis, Capillary↗
PubMed1998

Electrophoresis combined with novel mass spectrometry techniques: powerful tools for the analysis of proteins and proteomes.

Analytical and preparative electrophoresis separation techniques have been essential tools in protein biochemistry and the biological sciences in general. The combination of high resolution electrophoresis techniques with high performance analytical procedures has dramatically enhanced analytical protein biochemistry. In this report we describe the combination of electrophoretic separation techniques with electrospray ionization (ESI) tandem mass spectrometry (MS/MS). A series of different techniques, consisting of automated high performance liquid chromatography (HPLC)-MS/MS, capillary-HPLC-MS/MS, and solid phase extraction (SPE)-capillary zone electrophoresis (CZE)-MS/MS, are described in the context of the identification of high pmol to the low fmol amounts of proteins. Application of these powerful new tools for the analysis of proteins on a large proteome-wide scale is presented. Furthermore, the combination of orthogonal separation techniques, such as immobilized metal affinity chromatography (IMAC) with SPE-CZE-MS/MS, and IMAC followed by HPLC, and by SPE-CZE-MS/MS, are presented for the detailed investigation of post-translational modifications of specific proteins.

Amino Acid Sequence↗
PubMed1998

Proteome analysis: biological assay or data archive?

In this review we examine the current state of proteome analysis. There are three main issues discussed: why it is necessary to study proteomes; how proteomes can be analyzed with current technology; and how proteome analysis can be used to enhance biological research. We conclude that proteome analysis is an essential tool in the understanding of regulated biological systems. Current technology, while still mostly limited to the more abundant proteins, enables the use of proteome analysis both to establish databases of proteins present, and to perform biological assays involving measurement of multiple variables. We believe that the utility of proteome analysis in future biological research will continue to be enhanced by further improvements in analytical technology.

Animals↗
PubMed1998

Optimization of solid phase microextraction - capillary zone electrophoresis - mass spectrometry for high sensitivity protein identification.

We have previously described the use of a solid phase extraction (SPE) - capillary zone electrophoresis (CZE) - tandem mass spectrometry (MS/MS) system for protein analysis at the low femtomole to subfemtomole level. Here we describe the systematic optimization of a number of parameters which facilitate the use of the SPE-CZE-MS/MS system and further enhance its performance. Specifically, we describe a robust SPE cartridge design which can be assembled without the use of glue, the evaluation of procedures to chemically modify the inner wall of the fused-silica capillaries used in the system to improve separation and reproducibility, and the comparison of different reverse-phase (RP) resins used for the SPE cartridge. We also explored the effects of transient isotachophoresis with respect to system performance and compatibility with different fused-silica surface coatings, the RP resins used, and MS/MS. The enhanced performance of the optimized system is demonstrated by the analysis of calibrated tryptic digests of bovine serum albumin (BSA).

Acetonitriles↗
PubMed1997

A microfabricated device for rapid protein identification by microelectrospray ion trap mass spectrometry.

Nanoelectrospray mass spectrometry, the infusion at low flow rates of unseparated peptide mixtures representing protein proteolytic digests into an electrospray ionization mass spectrometer (MS), has been shown to be a suitable method for the analysis of small amounts of proteins. However, the current technique is time consuming, tedious, and difficult to automate. We used microfabrication technologies to construct a device for the sequential infusion of different peptide samples into an electrospray ionization MS without the need for sample manipulation. In this device, etched sample and buffer reservoirs are connected via etched channels to microelectrospray ion source. Peptide samples, typically unseparated tryptic digests of proteins, are applied to different reservoirs. A flow of liquid originating from a specific reservoir is generated and selectively directed toward the microsprayer and the MS by electroosmotic pumping. The analyte proteins are identified by searching sequence databases with the information contained in the collision-induced spectra of selected peptides. With this system, we have achieved a limit of detection in the low femtomoles per microliter range for peptide standards. We also show that samples deposited in different reservoirs can be sequentially mobilized without cross-contamination and that proteins can be conclusively identified at the low femtomoles per microliter level. The successful coupling online of microfabricated devices to an electrospray ionization MS represents an essential step toward the construction of automated, high-throughput, and high-sensitivity analytical systems.

Amino Acid Sequence↗
PubMed1997

Capillary electrophoresis of peptides and proteins at neutral pH in capillaries covalently coated with polyethyleneimine.

Two procedures for the derivatization of the inner wall of fused-silica capillaries for the analysis of peptides and proteins by capillary electrophoresis (CE) at neutral pH are presented. In the first procedure, polyethyleneimine (PEI) is covalently attached to the capillary wall. In the second procedure, PEI is additionally cross-linked. We present analysis of standard peptides and proteins by CE using the coated capillaries. These coatings will have application for the separation of protein complexes at neutral pH, prior to analysis by electrospray mass spectrometry.

Buffers↗
PubMed1997

Identification of proteins by capillary electrophoresis-tandem mass spectrometry. Evaluation of an on-line solid-phase extraction device.

Capillary electrophoresis-tandem mass spectrometry has been used successfully for the analysis of complex peptide mixtures. The method is limited by a relatively high concentration limit of detection and by matrix effects. Here we describe on-line coupling of a solid-phase microextraction device to a capillary electrophoresis-tandem mass spectrometry system. The performance of the integrated instrument was evaluated for the identification of proteins by their amino acid sequence. We report that the concentration limit of detection was improved at least 1000 fold to the low attomole/microliter range and that matrix effects were minimized by extensive sample clean-up during solid-phase extraction. We demonstrate that the implementation of a solid-phase extraction device significantly enhances capillary electrophoresis-tandem mass spectrometry as a method for the identification of low abundance proteins isolated from high-resolution two-dimensional polyacrylamide gels.

Amino Acid Sequence↗
PubMed1996

Pseudo-coulometric loading in capillary electrophoresis DNA sequencing.

While injection volumes in capillary electrophoresis are typically in the nanoliter range, it is difficult to physically prepare and manipulate samples much smaller than a microliter. As a result, only a small fraction of the analyte contained with the sample volume is transferred to the capillary. This problem is particularly acute in DNA sequencing applications, where on-column stacking is difficult and where the sequencing sample is relatively expensive to prepare. We report a method that transfers 75% of the DNA contained within a 3 microliters sample onto a capillary for DNA sequencing. This method relies on the use of very low ionic strength formamide to resuspend the DNA after an ethanol precipitation. The use of low ionic strength formamide achieves two tasks. First, it produces a very high resistance sample, which increases the voltage drop across the sample and decreases the field across the capillary. This electric field manipulation ensures that DNA fragments do not migrate down the capillary during the loading process, allowing long injection periods without excessive band-broadening. Second, the low ionic strength of the formamide increases the transference number of the DNA; more of the current passing through the injection tip of the capillary is carried by DNA fragments. In the limit of complete elimination of impurity ions from the loading solvent, current passing through the sample is carried only by DNA fragments and loading becomes a coulometric process.

DNA, Single-Stranded↗
PubMed1996

Protein identification by capillary zone electrophoresis/microelectrospray ionization-tandem mass spectrometry at the subfemtomole level.

A method for the identification of proteins by their amino acid sequence at the low-femtomole to subfemtomole sensitivity level is described. It is based on an integrated system consisting of a capillary zone electrophoresis (CZE) instrument coupled to an electrospray ionization triple- quadrupole tandem mass spectrometer (ESI-MS/MS) via a microspray interface. The method consists of proteolytic fragmentation of a protein, peptide separation by CZE, analysis of separated peptides by ESI-MS/MS, and identification of the protein by correlation of the collision-induced dissociation (CID) patterns of selected peptides with the CID patterns predicted from all the isobaric peptides in a sequence database. Using standard peptides applied to a 20-microns-i.d. capillary, we demonstrate an ESI-MS limit of detection of less than 300 amol and CID spectra suitable for searching sequence databases obtained with 600 amol of sample applied to the capillary. Successful protein identification by the method was demonstrated by applying 50 and 38 fmol of a tryptic digest of the proteins beta-lactoglobulin and bovine serum albumin, respectively, to the system.

Amino Acid Sequence↗
PubMed1996

Protein identification by solid phase microextraction-capillary zone electrophoresis-microelectrospray-tandem mass spectrometry.

We describe an analytical system for the rapid identification of proteins by correlation of tandem mass spectra with protein sequence databases. The system consists of an integrated solid phase microextraction/capillary zone electrophoresis peptide separation device that is connected through a microelectrospray ion source to a tandem mass spectrometer. The limits of detection are 660 amol of sample at a concentration limit of < 33 amol/microliters for peptide mass measurement, and < 10 fmol of sample, at a concentration limit of < 300 amol/microliters for peptide analysis by collision-induced dissociation. Using this system, we have identified low nanogram amounts of yeast proteins separated by high-resolution two-dimensional gel electrophoresis.

Databases, Factual↗
PubMed1994

Labeling of double-stranded DNA by ROX-dideoxycytosine triphosphate using terminal deoxynucleotidyl transferase and separation by capillary electrophoresis.

Terminal transferase is used to add a single fluorescently labeled dideoxynucleotide to double-stranded DNA prepared by restriction endonuclease action on a bacteriophage. The product is separated by capillary electrophoresis with both hydroxypropylmethylcellulose and non-cross-linked polyacrylamide. The reaction products generate single peaks for each fragment with hydroxypropylmethylcellulose. However, the higher resolution separation produced by non-cross-linked polyacrylamide shows that the product contains two components for each restriction digest fragment. This labeling technique should be useful in restriction fragment length polymorphism studies.

DNA↗
PubMed1994

Activation energy of single-stranded DNA moving through cross-linked polyacrylamide gels at 300 V/cm. Effect of temperature on sequencing rate in high-electric-field capillary gel electrophoresis.

In DNA sequencing, single-stranded DNA fragments are separated by gel electrophoresis. This separation is based on a sieving mechanism where DNA fragments are retarded as they pass through pores in the gel. In this paper, we present the mobility of DNA sequencing fragments as a function of temperature; mobility is determined in 4% T LongRanger gels at an electric field of 300 V/cm. The temperature dependence is compared with the predictions of the biased reptation model. The model predicts that the fragment length for the onset of biased reptation with stretching increases with the square of temperature; the data show that the onset of biased reptation with stretching decreases with temperature. Biased reptation fails to model accurately the temperature dependence of mobility. We analyzed the data and extracted the activation energy for passage of sequencing fragments through the gel. For fragments containing less than ca. 200 bases, the activation energy increases linearly with the number of bases at a rate of 25 J/mol per base; for longer fragments, the activation energy increases at a rate of 6.5 J/mol per base. This transition in the activation energy presumably reflects a change in conformation of the DNA fragments; small fragments exist in a random coil configuration and larger fragments migrate in an elongated configuration.

Chemical Phenomena↗
PubMed1994

Spatial and temporal depletion of ions from noncrosslinked denaturing polyacrylamide in capillary electrophoresis.

Electrical conductivity across a polyacrylamide-filled capillary decreases during the separation of DNA sequencing fragments. This conductivity decrease is localized to the first few centimeters at the injection (negative) end of the capillary; no conductivity change is noted at the detection (positive) end of the capillary. The zone of decreased conductivity extends further into the capillary as the separation proceeds. The zone is most important for freshly prepared capillaries; capillaries used nine days after polymerization generate an insignificant current drop. The data are consistent with ionic depletion due to differences in transport numbers between the separation medium and the buffer reservoirs.

Acrylic Resins↗
PubMedDate not supplied

High sensitivity identification of proteins by electrospray ionization tandem mass spectrometry: initial comparison between an ion trap mass spectrometer and a triple quadrupole mass spectrometer.

Recently, we have shown that a solid-phase-microextraction/capillary electrophoresis device coupled to an electrospray ionization triple quadrupole mass spectrometer through a microelectrospray interface represents a powerful analytical system for the rapid, conclusive and sensitive identification of proteins separated by gel electrophoresis. Here we report on the successful coupling of the same device to an electrospray ionization ion trap mass spectrometer and on the comparative evaluation of the performance of the triple quadrupole and ion-trap-based systems. In the ion trap mass spectrometer-based system, using a tryptic digest of a calibrated bovine serum albumin sample, we achieved limits of detection in the single mass spectrometry (MS) and tandem MS mode, respectively, of 400 amol (if 20 microL of solution at a concentration of 20 amol/microL was applied). The system was also successfully used to identify six yeast proteins isolated from a single analytical two-dimensional polyacrylamide gel. For the detection of unfragmented peptide ions both systems showed comparable sensitivity, whereas the ion-trap-based system showed superior performance with fragment ion spectra.

Amino Acid Sequence↗