Evaluation of packed capillary perfusion column HPLC/MS/MS for the rapid mapping and sequencing of enzymic digests

Kassel, Daniel B.; Shushan, Bori; Sakuma, Takeo; Salzmann, Jean Pierre.

doi:10.1021/ac00074a010

Anal. Chem.

1994

DOI: 10.1021/ac00074a010

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Evaluation of packed capillary perfusion column HPLC/MS/MS for the rapid mapping and sequencing of enzymic digests

Daniel B. Kassel¹,

Bori Shushan²,

Takeo Sakuma³

et al.

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Cited by 45 publications

(24 citation statements)

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“…Nowadays, capillary HPLC coupled with MS as a complementary method for two dimensional polyacrylamide gel electrophoresis has become an important tool for separating protein. [5][6][7] However, the front-end column technology need to be further improved.To date, there are mainly two kinds of capillary HPLC columns used. One is a packed column, typically filled with alkylated silica particles in fused-silica capillaries.…”

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Application of an in-situ Thermo-polymerized Porous Polymer: Creation of an On-column Frit for a Packed Capillary HPLC Column

Ding

et al. 2007

ANAL. SCI.

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Capillary high-performance liquid chromatography (capillary HPLC), [1][2][3][4] as an intermediate technique between conventional liquid chromatography and microchip separations, has been one of the main trends as a separation technique during the past decade.Especially, the development of proteomics and metabolomics has promoted the rapid development of capillary HPLC technology in recent years. Using capillary HPLC columns (typical size, 0.32 -0.075 mm i.d.) has many advantages over using a common HPLC column (4.6 mm i.d.). Miniaturization of the separation column has led to lower running costs and reduced environmental pollution by solvents and chemicals. For example, a reduction of the column i.d. from 4.6 to 0.32 mm allows the same analysis with more than 200-times less solvent as the mobile phase. On the other hand, capillary HPLC has low sample-volume requirements and easy connection to mass spectrometry (MS). These advantages are paid more attention in the field of life sciences, since bioanalytical applications are often sample-limited. Nowadays, capillary HPLC coupled with MS as a complementary method for two dimensional polyacrylamide gel electrophoresis has become an important tool for separating protein. [5][6][7] However, the front-end column technology need to be further improved.To date, there are mainly two kinds of capillary HPLC columns used. One is a packed column, typically filled with alkylated silica particles in fused-silica capillaries. The other is a monolithic column, 8,9 which is prepared by in-situ polymerization and avoids frit fabrication. Although monolithic capillary columns are prepared much more easily than packed capillary columns, packed capillary columns are mainly employed in proteomics research because of the availability of various packing materials. However, the packing of micronsized particles into a capillary tubing and the preparation of solid and stable on-column frits at the end of the columns are difficult techniques.Usually a capillary column needs tubing at the end of the column for connecting with the detector. The packed bed and the tubing for the detector are connected with a union or by glue. However, the connection can inevitably induce a dead volume. Considering the detrimental influence of the dead volume, the best design for the column is a zero dead volume connection between the packing bed and the tubing for the detector. If the packed bed and the tubing for the detector are integrated in one capillary, the detection window can be made very close to the frit, so as to obtain the minimum dead volume. In order to retain the packing material inside the fused-silica capillary, on-column frits have to be produced at the end of the packing bed.Several frit-making methods have been developed. Sintering a silica packing material 10 or together with sodium silicate 11 has often been used for end-frit making. However, a partially intense heating in the sintering process can destroy the structure of particles, leading to some active sites on the surface of the fri...

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mentioning

confidence: 99%

Application of an in-situ Thermo-polymerized Porous Polymer: Creation of an On-column Frit for a Packed Capillary HPLC Column

Ding

et al. 2007

ANAL. SCI.

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“…However, a more convenient and efficient on-line methodology, HPLC-MS, has become popular recently, where the ESI interface is the way of choice to directly analyze the eluate from an HPLC column with a mass spectrometer (7,(11)(12)(13)(14).…”

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confidence: 99%

Determination of the Limited Trypsinolysis Pathways of Tumor Necrosis Factor-α and Its Mutant by Electrospray Ionization Mass Spectrometry

Kim

Shin

Kim

et al. 1999

Analytical Biochemistry

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“…Kassel et al 5 coupled packed capillaries with mass spectrometry for fast protein analysis. In a related paper, Kassel and co-workers 6 reported the use of perfusion packed capillaries for peptide mapping. Using a 320 µm diameter packed capillary, they performed an analysis of 25 picomol of digested protein in 15 min.…”

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

MacNair

Opiteck

Jorgenson

et al. 1997

Rapid Commun. Mass Spectrom.

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Octadecyl-modified 1.5 µm diameter non-porous silica particles were packed in 150 µm i.d. (360 µm 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 µm diameter porous particles. The use of 1.5 µm diameter non-porous particles for reversed-phase liquid chromatography (RPLC) has been shown to increase resolution and reduce analysis time.1,2 High efficiency (N > 200 000 theoretical plates) separations of small organic analytes by isocratic RPLC require pressures of 1800 Bar (26 000 psi) using a 66 cm long capillary column packed with 1.5 µm diameter particles.1 Stationary phase supports of this size reduce analysis time by a factor of 3.3 over that for 5 µm diameter particles, at optimum chromatographic flow rates, using columns of the same length.This technology can be applied to difficult analytical problems including the analysis of mixtures of intact proteins and proteolytic digests. High resolution separations can be performed rapidly with this packing material in short, packed capillaries requiring moderate operating pressures. Coupling this RPLC technique with mass spectrometry creates a system well suited for identifying components in complex mixtures such as cell lysates and enzymatic digests with sensitivity on a par with conventional packed capillary LC/MS using 5 µm diameter porous particles.Perfusion column chromatography 3,4 is another approach to fast RPLC. Kassel et al.5 coupled packed capillaries with mass spectrometry for fast protein analysis. In a related paper, Kassel and co-workers 6 reported the use of perfusion packed capillaries for peptide mapping. Using a 320 µm diameter packed capillary, they performed an analysis of 25 picomol of digested protein in 15 min. These analyses are fast and show good resolution, but are not as sensitive as the more time-consuming separations using capillaries packed with 5 µm diameter particles. [7][8][9][10][11] . Considering that perfusion chromatography requires high mobilephase flow-rates, the analyte concentrations on-column are reduced. Since electrospray is concentration dependent, 12 the sensitivity suffers. In the work presented here, the sensitivity of capillaries packed with 5 µm diameter porous particles and the speed of perfusion chromatography were achieved simultaneously using 1.5 µm C 18 bonded nonporous silica particles. These particles were packed in 150 µm i.d. (360 µm o.d.) capillaries with lengths of 20 cm. Intact proteins and peptides generated from enzymatic digests were analyzed on these columns with mobile-phase gradients at pressures of 520 Bar (7500 psi). Peaks were identif...

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Evaluation of packed capillary perfusion column HPLC/MS/MS for the rapid mapping and sequencing of enzymic digests

Cited by 45 publications

References 6 publications

Application of an in-situ Thermo-polymerized Porous Polymer: Creation of an On-column Frit for a Packed Capillary HPLC Column

Application of an in-situ Thermo-polymerized Porous Polymer: Creation of an On-column Frit for a Packed Capillary HPLC Column

Determination of the Limited Trypsinolysis Pathways of Tumor Necrosis Factor-α and Its Mutant by Electrospray Ionization Mass Spectrometry

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

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