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

Abstract

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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“…The electrostatic potential at any point near the surface is related to the net number of electrical charges per unit volume p in the vicinity of the point by the Poisson's equation (2). Far away from the wall, the concentrations of positive and negative ions are equal.…”

Applications of mass spectrometry for the structural characterization of recombinant protein pharmaceuticals

“…To achieve comparable or improved resolution with shorter analysis times, RP columns with smaller particle size must be used in combination with an HPLC system capable of handling very high pressure. MacNair et al have shown that such a system can be constructed and demonstrated its use for the high efficiency separation of small molecules and protein digests (MacNair, Lewis, & Jorgenson, 1997a;MacNair et al, 1997b;MacNair, Patel, & Jorgenson, 1999). Tolley, Jorgenson, and Moseley, 2001 have demonstrated that high quality MS/MS spectra can be obtained for protein digests analyzed using a hybrid qTOF mass spectrometer coupled to an ultra high performance HPLC (UPLC) with a column packed with 1.5 mm sized particles.…”

Silica‐based monoliths for rapid peptide screening by capillary liquid chromatography hyphenated with electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry

“…The use of small spheres (1.5–3 μm) significantly enhances the separation efficiency15 under the limitation, however, of a drastically increased column back‐pressure 16. The capillaries packed with these particles (either porous or non‐porous) are capable of providing high separation efficiency in reasonably short elution times only if they are operated as very short beds or with expensive ultra‐high pressure LC equipment enabling system pressure drops between 700 and 1100 bar 17–19…”