High-Efficiency On-Line Solid-Phase Extraction Coupling to 15-150 um I.D. Column Liquid Chromatography for Proteomic Analysis

Journal Article · Tue Jul 15 00:00:00 EDT 2003 · Analytical Chemistry, 75(14):3596-3605

DOI:https://doi.org/10.1021/ac0300690· OSTI ID:15004583

Shen, Yufeng ^[1]; Moore, Ronald J ^[1]; Zhao, Rui ^[1]; Blonder, Josip ^[1]; Auberry, Deanna L ^[1]; Masselon, Christophe D ^[1]; Pasa Tolic, Ljiljana ^[1]; Hixson, Kim K ^[1]; Auberry, Kenneth J ^[1]; Smith, Richard D ^[1]

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1. BATTELLE (PACIFIC NW LAB)

Flexible manipulation of various properties of proteomic samples is important for proteomic analyses, but it has been little explored for newly developed approaches based on liquid chromatography (LC) in combination with mass spectrometry (MS). With miniaturization of the LC column inner diameter dimensions (required for improving the analysis sensitivity), this issue becomes more challenging due to the small flow rates and the increasing effects of extra column volume on the separation quality and its use for resolving complex proteomic mixtures. In this study, we used commercial switching valves (150-mm channels) to implement the on-line coupling of capillary LC columns with relatively large solid phase extraction (SPE) columns operated at 10,000 psi. With optimized column connections, switching modes, and SPE column dimensions, high-efficiency on-line SPE-capillary and nanoscale LC separations were obtained with peak capacities of~1000 for capillaries having inner diameters between 15 to 150 mm. The on-line coupled SPE columns increased the sample processing capabilities by~400-fold for sample solution volume and~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. Used with an ion trap tandem MS we could typically identify 1100-1500 peptides for analyses in a single 5-hour run. Peptides extracted on the SPE column and eluted from the LC column covered a hydrophilicity/hydrophobicity range that include an estimated~98% of all the tryptic peptides. The present implementation also facilitates automation and enables use of both disposable SPE columns and electrospray emitters, providing a robust basis for routine proteomic analyses.

• Research Organization: Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
• Sponsoring Organization: USDOE
• DOE Contract Number: AC06-76RL01830
• OSTI ID: 15004583
• Report Number(s): PNNL-SA-38207; ISSN 1520-6882; KP1102010; TRN: US1005191
• Journal Information: Analytical Chemistry, 75(14):3596-3605, Vol. 75, Issue 14; ISSN 0003-2700
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
72 PHYSICS OF ELEMENTARY PARTICLES AND FIELDS
99 GENERAL AND MISCELLANEOUS//MATHEMATICS, COMPUTING, AND INFORMATION SCIENCE
AUTOMATION
CAPILLARIES
CHROMATOGRAPHY
DIMENSIONS
FLOW RATE
IMPLEMENTATION
MASS SPECTROSCOPY
MEMBRANE PROTEINS
MINIATURIZATION
MIXTURES
PEPTIDES
PROCESSING
SENSITIVITY
SOLAR PROTONS
VALVES