Packed Capillary Reversed-Phase Liquid Chromatography with High-Performance Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry for Proteomics

Abstract

In this study, high-efficiency packed capillary reversed-phase liquid chromatography (RPLC) coupled on-line with high-performance Fourier transform ion cyclotron resonance (FTICR) mass spectrometry has been investigated for the characterization of complex cellular proteolytic digests. Long capillary columns (80-cm) packed with small (3-micron) C18 bonded particles provided a total peak capacity of approximately 1000 for cellular proteolytic polypeptides when interfaced with an ESI-FTICR mass spectrometer under composition gradient conditions at a pressure of 10,000 psi. Large quantities of cellular proteolytic digests (e.g., 500 micrograms) could be loaded onto packed capillaries of 150-micron inner diameter without a significant loss of separation efficiency. Precolumns with suitable inner diameters were found useful for improving the elution reproducibility without a significant loss of separation quality. Porous particle packed capillaries were found to provide better results than those containing nonporous particles because of their higher sample capacity. Two-dimensional analyses from the combination of packed capillary RPLC with high-resolution FTICR yield a combined capacity for separations of > 1 million polypeptide components and simultaneously provided information for the identification of the separated components based upon the accurate mass tag concept previously described.

Advances in proteomics data analysis and display using an accurate mass and time tag approach

“…This method benefits from high dynamic range, mass resolution and mass accuracy , Shen, et al, 2001b. Over the last decade we have developed and refined nanoscale capillary LC (nanoLC)-FTICR-MS approaches that have evolved into an accurate mass and time (AMT) tag approach (described below) that has proved to be a robust method for automated high-throughput proteomics studies (Belov, et al, 2004, Paša-Tolić et al, 2004.…”

Development and Evaluation of a Micro- and Nanoscale Proteomic Sample Preparation Method

“…The technical details and separation performance of this LC platform have been described previously. 28 In brief, the capillary column was made by slurry packing 3-μm Jupiter C 18 particles (Phenomenex, Torrence, CA) into a 65-cm length of 150 μm i.d. fused silica capillary (Polymicro Technologies Inc., Phoenix, AZ).…”

Peak Capacity Optimization of Peptide Separations in Reversed-Phase Gradient Elution Chromatography: Fixed Column Format

“…Since the time in which a solute can elute under gradient conditions actually extends from t 0 to t 0 + t D + t G ( t 0 is the column dead time, t D is the dwell time and t G is the gradient time), Snyder, 22 in contrast to Giddings and Horvath, estimated the time window as t G and thus approximated the maximum possible peak capacity as: (6) However, realizing that a real sample generally occupies only a fraction of the maximum possible time window (t G ), Snyder 22 returned to the use of a time window based on the first real and last real peak as initially formulated by Giddings and Horvath and then went on to define what he termed a "sample peak capacity" (n c **): (7) where t R,n and t R,1 are the same as t n and t 1 in eqs 1 and 2, respectively. Clearly, t G is the maximum possible time window that a real sample can occupy.…”