On-Column Sample Enrichment for Capillary Electrophoresis Sheathless Electrospray Ionization Mass Spectrometry: Evaluation for Peptide Analysis and Protein Identification (original) (raw)
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International Journal of Pharmaceutical Chemistry, 2021
The pharmaceutical industry and most particularly the biopharmaceutical industry are demanding advancements in analytical techniques. The field of proteomics and peptide mapping needs sensitive analysis and sophisticated instrumentation, which needs hyphenated analytical techniques to be explored. In addition to effective detection and characterization, Due to efficient and selective separation, predominant focus has been drawn to capillary electrophoresis/mass spectrometry (CE/MS) compared to widely used techniques such as slab-gel electrophoresis (SGE), liquid chromatography (LC) and capillary electrophoresis (CE) Several significant advances in CE/MS instrumentation and software have made CE/MS a competitive tool in the last decade to solve a wide variety of analytical and biopharmaceutical problems. This analysis discusses the technological aspects of CE / MS interfaces, analytical separation modes, MS analyzer, recent CE / MS approaches and applications specific to biopharmaceu...
Recent Developments in Capillary Electrophoresis–Mass Spectrometry of Proteins and Peptides
Analytical Sciences, 2005
Many researchers have invested considerable efforts toward improving capillary electrophoresis (CE)-mass spectrometry (MS) systems so they can be applied better to standard analyses. This review highlights the developments in CE-MS of proteins and peptides over the last five years. It includes the developments in interfaces, sample-enrichment techniques, microfabricated devices, and some applications, largely in capillary zone electrophoresis (CZE), capillary isoelectric focusing (CIEF) and capillary isotachophoresis formats.
Journal of Chromatography A, 2007
The use of solid-phase extraction coupled on-line to capillary electrophoresis using electrospray mass spectrometry detection (SPE-CE-ESI-MS) is described for the analysis of peptides in dilute solutions. A SPE microcartridge or analyte concentrator containing C 18 derivatized silica particles as the extraction sorbent was easily constructed near the inlet of the separation capillary using commercially available materials. The reversed-phase sorbent selectively retained the target peptides, enabling large volumes of the sample to be introduced (>100 L). The captured analytes were eluted in a small volume of an appropriate solution (20-50 nL). This resulted in sample clean-up and concentration enhancement, with minimum sample handling. As the SPE-CE conditions were compatible with on-line ESI-MS detection, the potential for identifying and characterizing the preconcentrated analytes by SPE-CE-ESI-MS using a sheath-flow CE-ESI-MS interface is also shown. Using separation electrolytes containing N-[carbamoylmethyl]-2-aminoethanesulfonic acid (ACES) at pH 7.4, an elution plug of 80:20 (v/v) (25 mM of formic acid in MeCN):H 2 O and a sheath liquid of 20 mM of acetic acid in 50:50 (v/v) methanol:H 2 O the concentration limits of detection for the analyzed peptides in the positive ion mode were lowered to nanogram per milliliter levels. The systematic optimization of the operational parameters involved in the development of the SPE-CE method is described in detail, in order to promote robust and quantitative SPE-CE-ESI-MS analysis and facilitate the widespread use of the technique.
ELECTROPHORESIS, 2003
High-efficiency peptide analysis using multimode pressure-assisted capillary electrochromatography/capillary electrophoresis (pCEC/pCE) monolithic polymeric columns and the separation of model peptide mixtures and protein digests by isocratic and gradient elution under an applied electric field with UV and electrospray ionization-mass spectrometry (ESI-MS) detection is demonstrated. Capillary multipurpose columns were prepared in silanized fused-silica capillaries of 50, 75, and 100 mm inner diameters by thermally induced in situ copolymerization of methacrylic monomers in the presence of n-propanol and formamide as porogens and azobisisobutyronitrile as initiator. N-Ethylbutylamine was used to modify the chromatographic surface of the monolith from neutral to cationic. Monolithic columns were termed as multipurpose or multimode columns because they showed mixed modes of separation mechanisms under different conditions. Anion-exchange separation ability in the liquid chromatography (LC) mode can be determined by the cationic chromatographic surface of the monolith. At acidic pH and high voltage across the column, the monolithic stationary phase provided conditions for predominantly capillary electrophoretic migration of peptides. At basic pH and electric field across the column, enhanced chromatographic retention of peptides on monolithic capillary column made CEC mechanisms of migration responsible for separation. The role of pressure, ionic strength, pH, and organic content of the mobile phase on chromatographic performance was investigated. High efficiencies (exceeding 300 000 plates/m) of the monolithic columns for peptide separations are shown using volatile and nonvolatile, acidic and basic buffers. Good reproducibility and robustness of isocratic and gradient elution pressure-assisted CEC/CE separations were achieved for both UV and ESI-MS detection. Manipulation of the electric field and gradient conditions allowed highthroughput analysis of complex peptide mixtures. A simple design of sheathless electrospray emitter provided effective and robust low dead volume interfacing of monolithic multimode columns with ESI-MS. Gradient elution pressure-assisted mixed-mode separation CE/CEC-ESI-MS mass fingerprinting and data-dependent pCE/pCEC-ESI-MS/MS analysis of a bovine serum albumin (BSA) tryptic digest in less than 5 min yielding high sequence coverage (73%) demonstrated the potential of the method.
Top-down analysis of basic proteins by microchip capillary electrophoresis mass spectrometry
Rapid Communications in Mass Spectrometry, 2006
A system of microchip capillary electrophoresis/electrospray ionization mass spectrometry (mchip-CE/ESI-MS) for rapid characterization of proteins has been developed. Capillary electrophoresis (CE) enables rapid analysis of a sample present in very small quantity, such as at femtomole levels, at high resolution. Faster CE/MS analysis is expected by downsizing the normal capillary to the microchip (mchip) capillary. Although rapidity and high resolution are advantages of CE separation, electroosmotic flow (EOF) instability caused by the interaction between proteins and the microchannel surface results in low reproducibility in the analysis of basic proteins under neutral pH conditions. By coating the microchannel surface with a basic polymer, polyE-323, basic proteins, which have pI values of over 7.5, could be separated and detected by mchip-CE/MS on quadrupole (Q) and time-offlight (TOF) hybrid instruments. By increasing the cone and collision voltages during the analysis by mchip-CE/ESI-MS of a small protein, some product ions, which contain the sequence information, could also be obtained, i.e., 'top-down' analysis of the protein could be accomplished with this mchip-CE/MS system. To our knowledge, this is the first report of 'top-down' analysis of a protein by mchip-CE/MS. Since it requires a much shorter time and a smaller sample amount for analysis than the conventional liquid chromatography (LC)/ESI-MS method, mchip-CE/MS promises to be suitable for the high-throughput characterization of proteins.
Journal of Chromatography A, 2007
To simplify capillary electrophoresis-mass spectrometry (CE-MS) operation, a background electrolyte (BGE) containing a polymer additive is introduced that allows the analysis of peptides and protein mixtures in underivatized fused-silica capillaries without any pretreatment, thereby increasing throughput. The most important characteristic of these polymer additives is that they do not significantly suppress the signals of the proteins and peptides under electrospray ionization, thereby allowing them to be used as an additive to common BGEs that are used for CE-MS analysis of peptide and protein mixtures. In addition, because the fused-silica capillary inner wall is continuously coated with the polymer additive, migration irreproducibility, due to the degradation of the capillary inner wall coating, under CE-MS is minimized. High sensitivity of detection, migration reproducibility, and ease of fabrication allow CE-MS analyses that require long analysis time, such as (CE-MS/MS) n , to be performed with ease. The utility of this background electrolyte has been demonstrated for the analysis of complex protein digests and intact proteins.
Journal of the American Society for Mass Spectrometry, 1998
An in-capillary electrode sheathless interface was applied to the capillary electrophoresis/ electrospray ionization-mass spectrometry (CE/ESI-MS) analysis of mixtures of small peptides, proteins, and tryptic digests of proteins. The effects of different experimental parameters on the performance of this CE/ESI-MS interface were studied. The distance of the in-capillary electrode from the CE outlet and the length of the electrode inside the capillary had no significant effects on the CE separation and ESI behavior under the experimental conditions used. However, significant enhancement of the sensitivity resulted from the use of narrower CE capillaries. Using a quadrupole mass spectrometer, an aminopropylsilane-coated capillary, and a wide scan mass-to-charge ratio range of 500 -1400, detection limits of approximately 4, 1, and 0.6 fmol for cytochrome c and myoglobin were achieved for 75-, 50-, and 30-m inner diameter capillaries, respectively. Approximately one order of magnitude lower detection limits were achieved under the multiple-ion monitoring mode. The application of the in-capillary electrode sheathless interface to real-world samples was demonstrated by CE/ ESI-MS analysis of a human blood sample
On-column sample enrichment for the high-sensitivity sheath-flow CE-MS analysis of peptides
Analytical and Bioanalytical Chemistry, 2006
A sample enrichment technique to increase sensitivity in capillary electrophoresis-mass spectrometry (CE-MS) is described. Peptides or glycopeptides are retained and concentrated on a short (3-5-mm) reversedphase (C18) packed-bed situated in the fused-silica separation capillary and are subsequently released for electrophoretic separation by injection of an organic elutant. The concentration limits of detection are in the high picomolar range with a sheath-flow CE-MS interface.
On-line capillary electrophoresis-mass spectrometry for the analysis of biomolecules
Electrophoresis, 2004
Mass spectrometry (MS) has become a key tool for the characterization of biologically relevant molecules in the last decade. Due to the complexity of most biological samples an upstream separation is essential. Capillary electrophoresis (CE) has gained much interest due to its high separation efficiency, speed, and often complementary selectivity to liquid chromatography. We describe the state-of-the-art of on-line CE-MS for the analysis of molecules of biological origin. The characterization of peptides, including the study of post-translational modifications, intact proteins, oligonucleotides, and related interaction studies are reviewed. Relevant publications are summarized in tables, including some important method parameters. Key applications are discussed with respect to the advantages and limitations of CE-MS. Coupling interfaces, preconcentration techniques, capillary coatings, and the different CE techniques, e.g., capillary zone electrophoresis, capillary isoelectric focusing, capillary gel electrophoresis, etc. are briefly discussed against the background of their bioanalytical applications.