Introducing Samples Directly into Electrospray Ionization Mass Spectrometers Using Microscale Capillary Liquid Chromatography

Terry D. Lee1, Roger E. Moore1, Mary K. Young1

1 Beckman Research Institute of the City of Hope, Duarte
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 16.9
DOI:  10.1002/0471140864.ps1609s22
Online Posting Date:  May, 2001
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Abstract

This unit describes the design and operation of a microscale electrospray (ES) interface suitable for the on‐line liquid chromatography (LC) separation and mass spectrometry (MS) analysis of mixtures of peptides and proteins. The interface utilizes an ES needle packed with reversed‐phase support. Such a design has the advantage of minimizing any void volume between the end of the column and point of electrospray ionization, thus maintaining the integrity of the LC separation and maximizing sensitivity. Here, protocols are presented for construction of an integrated LC column ES needle in‐house, packing the ES needle, and mounting and using the microscale ES LC/MS interface assembly. Various options for low‐flow solvent delivery systems are also discussed.

     
 
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Table of Contents

  • Basic Protocol 1: Building an Integrated LC Column ES Needle
  • Basic Protocol 2: Packing the ES Needle
  • Basic Protocol 3: Mounting and Using the Microscale ES LC/MS Interface Assembly
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Building an Integrated LC Column ES Needle

  Materials
  • 360‐µm‐o.d. × 150‐µm‐i.d. fused silica capillary (ES needle; Polymicro Technology)
  • Laser micopipet puller (e.g., Sutter Instruments)
  • 140‐µm‐o.d. × 25‐µm‐i.d. fused silica capillary (Polymicro Technology)
  • Tool for cleaving fused silica capillaries
  • Low‐power dissecting microscope
  • GF/A filter paper (Whatman)

Basic Protocol 2: Packing the ES Needle

  Materials
  • 5‐ to 10‐µm particle size silica‐based reversed‐phase chromatography medium
  • Acetonitrile
  • Pulled fused silica capillary needle with frit (column/ES needle, see protocol 1)
  • Empty columns with fritted tips, 360‐µm‐o.d. × 75‐µm‐i.d., 8‐µm orifice (optional; can be obtained commercially from New Objective)
  • 1/16‐ × 1/32‐in. stainless steel reducing union
  • 1/32‐in. to 0.4‐mm graphite reducing ferrule
  • 1/16‐in. gauge plug
  • Packing reservoir: 1/16‐in.‐o.d. × 0.02‐in.‐i.d. × 10‐cm long stainless steel tubing with a compression screw and metal ferrule permanently attached to each end
  • 1/16 ‐in. stainless steel union
  • 1‐ml disposable syringe and 16‐G blunt‐ended needle
  • Compression screws
  • 1.5‐ml microcentrifuge tubes
  • High pressure pump

Basic Protocol 3: Mounting and Using the Microscale ES LC/MS Interface Assembly

  Materials
  • Buffer A
  • Cytochrome c tryptic digest mixture test sample
  • Liquid chromatography (LC) pump
  • PEEK micro Tee (Upchurch)
  • PEEK sleeve (Upchurch)
  • Packed fused silica ES needle (see protocol 2; also available commercially from New Objective)
  • 0.025‐in.‐diameter gold wire (Alfa Aesar)
  • ES mass spectrometer and alligator clip
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Figures

Videos

Literature Cited

Literature Cited
   Chervet, J.P., Meijvogel, C.J., Ursem, M., and Salzmann, J.P. 1992. Recent advances in capillary liquid chromatography. LC‐GC. 10: 140‐148.
   Chervet, J.P., Ursem, M., and Salzmann, J.B. 1996. Instrumental requirements for nanoscale liquid chromatography. Anal. Chem. 68: 1507‐1512.
   Davis, M.T. and Lee, T.D. 1998. Rapid protein identification using a microscale electrospray LC/MS system on an ion trap mass spectrometer. J. Am. Soc. Mass Spectrom. 9: 194‐201.
   Davis, M.T., Stahl, D.C., and Lee, T.D. 1995. Low flow high‐performance liquid chromatography solvent delivery system designed for tandem capillary liquid chromatography mass spectrometry. J. Am. Soc. Mass Spectrom. 6: 571‐577.
   Ducret, A., Bartone, N., Haynes, P.A., Blanchard, A., and Aebersold, R. 1998. A simplified gradient solvent delivery system for capillary liquid chromatography electrospray ionization mass spectrometry. Anal. Biochem. 265: 129‐138.
   Gatlin, C.L., Kleemann, G.R., Hays, L.G., Link, A.J., and Yates, J.R. III. 1998. Protein identification at the low femtomole level from silver stained gels using a new fritless electrospray interface for liquid chromatography‐microspray and nanospray mass spectrometry. Anal. Biochem. 263: 93‐101.
   Moore, R.E., Licklider, L., Schumann, D., and Lee, T.D. 1998. A micro‐scale electrospray interface incorporating a monolithic, polystyrene‐divinylbenzene support for on‐line liquid chromatography‐tandem mass spectrometry analysis of peptides and proteins. Anal. Chem. 70: 4879‐4884.
Internet Resources
   http://www.cityofhope.org/microseq/download.html
  Web site for downloading information on custom built electrospray interfaces and low flow rate gradient pumping systems.
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