High-sensitivity peptide mapping using packed-capillary liquid chromatography and electrospray ionization mass spectrometry

Banks, J. Fred

doi:10.1016/0021-9673(96)00319-6

Cited by 42 publications

(21 citation statements)

References 25 publications

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“…Since bioanalytical applications are often sample-limited, and miniaturization of the separation column promises to enhance the detection sensitivity because of lower dilution effects [1], micro-high performance liquid chromatography (micro-HPLC) technology has been developed rapidly in recent years. Mass spectrometry (MS) is an ideal highly sensitive detector, and has been coupled with micro-HPLC through an electrospray ionization (ESI) interface to obtain high sensitivity analysis of peptide mixtures [2], a so-called "bottom-up" approach in proteome research. But for proteins, due to the difficulty of high resolution separation, direct analysis of proteinaceous analytes by HPLC/ESI-MS is still under development [3 -4].…”

Section: Introductionmentioning

confidence: 99%

Preparation and characterization of alkylated polymethacrylate monolithic columns for micro‐HPLC of proteins

Zhang²,

Xiang

et al. 2004

J of Separation Science

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Protein separations were carried out by micro-high performance liquid chromatography (micro-HPLC) with surface alkylated monolithic columns, which were prepared by in situ copolymerization of glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EGDMA) in the presence of dodecanol and toluene as porogens. First, glycidyl groups at the surface of the porous monolith were hydrolyzed with sulfuric acid. The hydroxyl groups thus formed were then reacted with n-alkyl chloride to form alkylated stationary phase. Separation performance for proteins on columns with C18 and C8 stationary phases was compared. The results showed that a poly(GMA-EGDMA) support derivatized with octadecyl moieties could achieve much better resolution than one with octyl groups. A protein mixture was separated with the octadecylated poly-(GMA-EGDMA) monolithic column, and the effluent peaks were collected and analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The physical properties of the monolithic columns such as column morphology, surface area, mesopore size distribution, and column permeability were further characterized by scanning electron microscopy (SEM), multipoint BET nitrogen adsorption/desorption, and Darcy's law, respectively.

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Section: Introductionmentioning

confidence: 99%

Preparation and characterization of alkylated polymethacrylate monolithic columns for micro‐HPLC of proteins

Zhang²,

Xiang

et al. 2004

J of Separation Science

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“…Peptide mapping is a powerful approach to identify and characterize proteins [1][2][3][4]. The protein of interest is digested by a proteolytic enzyme, typically trypsin, and the resulting peptide fragments are separated and then identified by mass spectrometry.…”

Section: Introductionmentioning

confidence: 99%

On‐line protein digestion and peptide mapping by capillary electrophoresis with post‐column labeling for laser‐induced fluorescence detection

Schoenherr

et al. 2004

Electrophoresis

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A nanoliter enzyme microreactor was developed for on-line capillary electrophoresis (CE) peptide mapping of proteins, allowing picomole quantities of proteins to be digested. The enzyme microreactor was formed by immobilizing trypsin onto a monolithic capillary column, which was prepared by in situ polymerization of glycidyl methacrylate and ethylene dimethacrylate in a capillary. Highly efficient digestion of three protein standards was demonstrated. The detection of peptide fragments in CE was enhanced by post-column derivatization and laser-induced fluorescence detection. The microreactor has a volume of about 30 nL and is coupled with a separation capillary via a fluid joint for on-line digestion. The overall analysis, including digestion and separation, lasted only about 16 min. Column efficiencies > 300 000 plates/m were obtained for most peaks in the electropherogram of an on-line peptide mapping experiment of denatured alpha-lactalbumin under optimal conditions.

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“…Various studies on the comparison of miniaturized LC-MS and standard LC-MS configurations using isocratic or gradient elution have been reported [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15]. Several groups only used LC columns of different ID and made no adaptations to the LC equipment and interface setup when the flow rate was changed to study the effect on the sensitivity [2,3,5,[7][8][9][10][11][12]. In such cases, miniaturized LC columns were tested in systems with relatively large dead volumes and suboptimal linear velocities of the mobile phase in the ESI interface.…”

Section: Introductionmentioning

confidence: 99%

Evaluation of the sensitivity of miniaturized liquid chromatography‐electrospray ionization‐mass spectrometry for pharmaceutical analysis

Kranendijk¹,

Waterval

Somsen

et al. 2005

J of Separation Science

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LC-ESI-MS is applied frequently in pharmaceutical analysis. The sample amount is generally not restricted, however with LC-ESI-MS, a lack of sensitivity may still be observed with standard-bore LC columns in isocratic mode. Therefore, it was investigated whether increased sensitivity could be achieved by using miniaturized LC-ESI-MS. Seven columns ranging from 0.1 to 4.6 mm ID were tested using several instrument setups. For proper comparison, a sensitivity gain factor (SGF) was introduced. The SGF expresses the extra sensitivity that may be obtained on top of the normal increase of peak concentration, which can be expected when the column ID is reduced. Desogestrel, mirtazapine, and sugammadex sodium were used as test compounds. For desogestrel and sugammadex sodium, the SGF increased up to a factor of 5-13 when the column ID was reduced, indicating enhanced ionization efficiencies at lower flow rates. Optimum sensitivity was found for the 0.3 mm column coupled in combination with a microinjection valve and a dedicated low flow rate interface. For mirtazapine, no increase of SGF was observed when the column ID was decreased. Apparently, the ionization efficiency of this compound is not affected by the flow rate and the spray quality.

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High-sensitivity peptide mapping using packed-capillary liquid chromatography and electrospray ionization mass spectrometry

Cited by 42 publications

References 25 publications

Preparation and characterization of alkylated polymethacrylate monolithic columns for micro‐HPLC of proteins

Preparation and characterization of alkylated polymethacrylate monolithic columns for micro‐HPLC of proteins

On‐line protein digestion and peptide mapping by capillary electrophoresis with post‐column labeling for laser‐induced fluorescence detection

Evaluation of the sensitivity of miniaturized liquid chromatography‐electrospray ionization‐mass spectrometry for pharmaceutical analysis

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