High-Efficiency On-Line Solid-Phase Extraction Coupling to 15−150-μm-i.d. Column Liquid Chromatography for Proteomic Analysis

Abstract

The ability to manipulate and effectively utilize small proteomic samples is important for analyses using liquid chromatography (LC) in combination with mass spectrometry (MS) and becomes more challenging for very low flow rates due to extra column volume effects on separation quality. Here we report on the use of commercial switching valves (150-microm channels) for implementing the on-line coupling of capillary LC columns operated at 10,000 psi with relatively large solid-phase extraction (SPE) columns. With the use of optimized column connections, switching modes, and SPE column dimensions, high-efficiency on-line SPE-capillary and nanoscale LC separations were obtained demonstrating peak capacities of approximately 1000 for capillaries having inner diameters between 15 and 150 microm. The on-line coupled SPE columns increased the sample processing capacity by approximately 400-fold for sample solution volume and approximately 10-fold for sample mass. The proteomic applications of this on-line SPE-capillary LC system were evaluated for analysis of both soluble and membrane protein tryptic digests. Using an ion trap tandem MS it was typically feasible to identify 1100-1500 unique peptides in a 5-h analysis. Peptides extracted from the SPE column and then eluted from the LC column covered a hydrophilicity/hydrophobicity range that included an estimated approximately 98% of all tryptic peptides. The SPE-capillary LC implementation also facilitates automation and enables use of both disposable SPE columns and electrospray emitters, providing a robust basis for automated proteomic analyses.

Charge competition and the linear dynamic range of detection in electrospray ionization mass spectrometry

“…This shows that the upper concentration limit of the LC-ESI-MS dynamic range can be extended by using a larger diameter LC column at a given sample mass loading and the same separation efficiency (i.e., constant V l and L p ). However, the apparent benefit disappears when sample size is scaled with flow rate, and it should be noted that a smaller diameter column will always provide higher ionization efficiency and be attractive for cases where sample size is limited [28,29].…”

“…It is expected that lower flow rate nano-electrosprays will approach a regime where ionization efficiencies will become similar for any compound, where analytes compete effectively with solvent related species for charge, and ionization efficiencies will approach 100%. While operation under such conditions poses challenges for both the separations and MS sensitivity, recent results have demonstrated the feasibility of such approaches for ultra-sensitive and broad dynamic range studies of complex proteome samples [28,29].…”

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

“…If we assume that the peak width in gradient elution is approximately independent of retention, 21 integration of eq 3 gives: (5) where W is the fixed peak width. By his use of t A , not the time of the first peak as per Giddings, it is evident that Grushka modified the peak capacity concept although he did not comment on the difference between his equation and Giddings'.…”

Targeted tandem mass spectrometry for high-throughput comparative proteomics employing NanoLC-FTICR MS with external ion dissociation

“…Peptide reversed phase liquid chromatography (RPLC) separations were performed using a system similar to that°described°by°Shen°et°al.° [11].°The°separation°was performed under constant pressure conditions at 10,000 psi, using two ISCO (Lincoln, NE) model 100 DM high-pressure syringe pumps. The column (80 cm ϫ 50 m) was packed in-house with Phenomenex (Torrance, CA) Jupiter particles (C18 stationary phase; 3-m particles; 300 Å pore size).…”