skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: MicroSPE-nanoLC-ESI-MS/MS Using 10-μm-i.d. Silica-Based Monolithic Columns for Proteomics

Abstract

Silica-based monolithic narrow bore capillary columns (25 cm x 10 µm i.d.) with an integrated nanoESI emitter has been developed to provide high quality and robust microSPE-nanoLC-ESI-MS analyses. The integrated nanoESI emitter adds no dead volume to the LC separation, allowing stable electrospray performance to be obtained at flow rates of ~10 nL/min. In an initial application we identified 5510 unique peptides covering 1443 distinct Shewanella oneidensis proteins from a 300 ng tryptic digest sample in a single 4-h LC-MS/MS analysis using a linear ion trap MS (LTQ). We found the use of an integrated monolithic ESI emitter provided enhanced resistance to clogging and good run-to-run reproducibility.

Authors:
 [1];  [1];  [1];  [1]
  1. Pacific Northwest National Lab. (PNNL), Richland, WA (United States). Biological Sciences Div.
Publication Date:
Research Org.:
Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
898092
Report Number(s):
PNNL-SA-49661
Journal ID: ISSN 0003-2700; 400412000
DOE Contract Number:
AC05-76RL01830
Resource Type:
Journal Article
Resource Relation:
Journal Name: Analytical Chemistry; Journal Volume: 79; Journal Issue: 2
Country of Publication:
United States
Language:
English
Subject:
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; Proteomics, MicroSPE-nanoLC-ESI-MS, Silica-based monolithic column, integrated monolithic ESI emitter, cone-jet electrospray.

Citation Formats

Luo, Quanzhou, Page, Jason S., Tang, Keqi, and Smith, Richard D. MicroSPE-nanoLC-ESI-MS/MS Using 10-μm-i.d. Silica-Based Monolithic Columns for Proteomics. United States: N. p., 2007. Web. doi:10.1021/ac061603h.
Luo, Quanzhou, Page, Jason S., Tang, Keqi, & Smith, Richard D. MicroSPE-nanoLC-ESI-MS/MS Using 10-μm-i.d. Silica-Based Monolithic Columns for Proteomics. United States. doi:10.1021/ac061603h.
Luo, Quanzhou, Page, Jason S., Tang, Keqi, and Smith, Richard D. Mon . "MicroSPE-nanoLC-ESI-MS/MS Using 10-μm-i.d. Silica-Based Monolithic Columns for Proteomics". United States. doi:10.1021/ac061603h.
@article{osti_898092,
title = {MicroSPE-nanoLC-ESI-MS/MS Using 10-μm-i.d. Silica-Based Monolithic Columns for Proteomics},
author = {Luo, Quanzhou and Page, Jason S. and Tang, Keqi and Smith, Richard D.},
abstractNote = {Silica-based monolithic narrow bore capillary columns (25 cm x 10 µm i.d.) with an integrated nanoESI emitter has been developed to provide high quality and robust microSPE-nanoLC-ESI-MS analyses. The integrated nanoESI emitter adds no dead volume to the LC separation, allowing stable electrospray performance to be obtained at flow rates of ~10 nL/min. In an initial application we identified 5510 unique peptides covering 1443 distinct Shewanella oneidensis proteins from a 300 ng tryptic digest sample in a single 4-h LC-MS/MS analysis using a linear ion trap MS (LTQ). We found the use of an integrated monolithic ESI emitter provided enhanced resistance to clogging and good run-to-run reproducibility.},
doi = {10.1021/ac061603h},
journal = {Analytical Chemistry},
number = 2,
volume = 79,
place = {United States},
year = {Mon Jan 01 00:00:00 EST 2007},
month = {Mon Jan 01 00:00:00 EST 2007}
}
  • We report on the preparation and performance of a high efficiency 70 cm ´ 20 µm i.d. silica-based monolithic capillary column. With a mobile phase delivery pressure of 5000 psi, this monolithic column provides flow rates as low as ~40 nL/min at an LC linear velocity of ~0.24 cm/s. The resultant columns provided a separation peak capacity of ~420 under conditions of on-line coupling micro solid phase extraction (SPE) and nanoelectrospray ionization (ESI) mass spectrometry (MS) for a Shewanella oneidensis tryptic digest. A sensitivity of ~15 attomole for detection of peptides was obtained when a conventional ion trap MS/MS was usedmore » for the detection. The sensitivity and separation efficiency of this column enabled identification of 2367 different peptides from 855 S. oneidensis distinct proteins from a 2.5 µg tryptic digest sample in a single 10-h analysis by nanoLC/MS/MS. The run-to-run and column-to-column reproducibility was investigated for proteomic analyses.« less
  • New approaches for ultra-sensitive proteomics are described for the characterization of complex protein (proteomic) samples of&< 50 ng total mass. Ultra-high sensitivity was achieved using high-efficiency 15-m i.d. capillary liquid chromatography (i.e. nanoLC) coupled on-line to a high-sensitivity Fourier transform ion cyclotron resonance (FTICR) mass spectrometer (MS) through a nanoscale electrospray ionization (nanoESI) interface. The high separation efficiency (peak capacities of {approx}103 with average peak widths of {approx}15 s) and small mobile phase flow rates ({approx}20 nL/min at optimal linear velocities of {approx}0.2 cm/s) from the nanoLC and the resulting high ionization efficiency of the nanoESI provided confident protein identificationmore » from < 75-zeptomole of individual proteins (e.g. with 6 tryptic peptides from albumin) and an estimated {approx}10 zeptomole ({approx}6000 molecules) sensitivity for peptide detection. Application of the nanoLC with ion trap MS/MS also allowed targeted protein identification at low attomole levels. The on-line coupled micro solid phase extraction allowed loading of sample solutions at 8 L/min, and provided a 250 attomolar peptide concentration detection limit using FTICR MS. This sensitivity enabled identification of proteins from 0.5 pg of a whole proteome extract tryptic digest sample. The proteome measurement dynamic range, protein identification overlap, and proteome quantitation accuracy were also investigated. An modified accurate mass and time tag data analysis methodology was used for peptide and protein identification, allowing the nanoLC-FTICR MS approach to identify 872 proteins from a 3 hour analysis of a 2.5 ng Deinococcus radiodurans proteome sample. The zeptomole level sensitivity provides a basis for extension of proteomics studies to low numbers of cells, and potentially a single mammalian cell.« less
  • We describe approaches for proteomics analysis using electrospray ionization-tandem mass spectrometry coupled with fast reversed-phase liquid chromatography (RPLC) separations. The RPLC separations used 50 µm i.d. fused silica capillaries packed with micron-sized C18-bonded porous silica particles and achieved peak capacities of 130-420 for peptides. When these separations were combined with a linear ion trap mass spectrometer, ~1,000 proteins could be identified in 50 minutes based upon the identification of ~4,000 tryptic peptides; ~550 proteins in 20 minutes from ~1,800 peptides; and ~250 proteins in 8 minutes from ~700 peptides for a S. oneidensis tryptic digest. The dynamic range for proteinmore » identification was determined to be ~3-4 orders magnitude of relative protein abundance on the basis of known proteins in human blood plasma analyses. We found that 55% of the MS/MS spectra acquired during the entire analysis (and up to 100% of the MS/MS spectra acquired from the most data rich zone) had sufficient quality for identifying peptides. The results indicate that such analyses using very fast (minutes) RPLC separations based on columns packed with micro-sized porous particles are primarily limited by the MS/MS analysis speed.« less
  • ABSTRACT-Targeted tandem mass spectrometry (MS/MS) is an attractive proteomic approach that allows selective identification of peptides exhibiting abundance differences between culture conditions and/or diseased states. Herein, we report on a targeted LC-MS/MS capability realized with a 7 Tesla Fourier transform ion cyclotron resonance (FTICR) mass spectrometer equipped with a quadrupole interface that provides data-dependent ion selection, accumulation, and dissociation externally to the ICR trap. Identification of a subset of differentially abundant proteins from Shewanella oneidensis grown under suboxic vs. aerobic conditions demonstrates the feasibility of such approach. High mass resolution offered by FTICR and effective on-the-fly elution time correction facilitatedmore » accurate selection of targets, while high mass measurement accuracy MS/MS data resulted in unambiguous peptide identifications.« less
  • Described here are the results from the profiling of the proteins arginine vasopressin (AVP) and adrenocorticotropic hormone (ACTH) from normal human pituitary gland and pituitary adenoma tissue sections using a fully automated droplet-based liquid microjunction surface sampling-HPLC-ESI-MS/MS system for spatially resolved sampling, HPLC separation, and mass spectral detection. Excellent correlation was found between the protein distribution data obtained with this droplet-based liquid microjunction surface sampling-HPLC-ESI-MS/MS system and those data obtained with matrix assisted laser desorption ionization (MALDI) chemical imaging analyses of serial sections of the same tissue. The protein distributions correlated with the visible anatomic pattern of the pituitary gland.more » AVP was most abundant in the posterior pituitary gland region (neurohypophysis) and ATCH was dominant in the anterior pituitary gland region (adenohypophysis). The relative amounts of AVP and ACTH sampled from a series of ACTH secreting and non-secreting pituitary adenomas correlated with histopathological evaluation. ACTH was readily detected at significantly higher levels in regions of ACTH secreting adenomas and in normal anterior adenohypophysis compared to non-secreting adenoma and neurohypophysis. AVP was mostly detected in normal neurohypophysis as anticipated. This work demonstrates that a fully automated droplet-based liquid microjunction surface sampling system coupled to HPLC-ESI-MS/MS can be readily used for spatially resolved sampling, separation, detection, and semi-quantitation of physiologically-relevant peptide and protein hormones, such as AVP and ACTH, directly from human tissue. In addition, the relative simplicity, rapidity and specificity of the current methodology support the potential of this basic technology with further advancement for assisting surgical decision-making.« less