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. 1998 Oct 1;263(1):93-101. doi: 10.1006/abio.1998.2809.

Abstract

Conventional capillary liquid chromatography/mass spectrometry (LC/MS) typically employs low microl/min flow rates with gas/liquid sheath to enhance spray stability. Over the past several years a number of reports have demonstrated success with electrospray (ES) interface designs optimized for submicroliter/min flows which have clear advantages in terms of enhancement of detection limit, lower sample consumption, and ability to accommodate a wider range of buffer conditions. We report here a fritless electrospray interface (FESI) design that is inexpensive and robust and can be operated and adapted to accommodate a variety of applications for submicroliter/min flow rates. The novelty of this interface revolves around the use of a fritless microcapillary column and precolumn application of electrospray voltage at a microtee junction to achieve stable microspray and nanospray flow rates. This sheathless FESI device eliminates postcolumn dead volume since small particles (</= 10 micron) are packed directly into laser-pulled fused silica capillary needles from which a spray originates. For analysis of proteins/peptides in solution, low femtomole sensitivity has been achieved (attomoles for selected-ion monitoring), while low nanogram sensitivity was attained for proteins derived from in-gel-digested silver-stained bands from 1-D and 2-D gels. Several applications for tandem MS protein/peptide identification using LC-microspray, LC-nanospray, or infusion nanospray are presented.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Chromatography, Liquid / instrumentation
  • Chromatography, Liquid / methods*
  • Electrophoresis, Gel, Two-Dimensional
  • Electrophoresis, Polyacrylamide Gel
  • Fungal Proteins / analysis
  • Fungal Proteins / chemistry
  • Mass Spectrometry / instrumentation
  • Mass Spectrometry / methods*
  • Molecular Sequence Data
  • Peptides / analysis
  • Peptides / chemistry
  • Proteins / analysis*
  • Proteins / chemistry
  • Saccharomyces cerevisiae / chemistry
  • Sensitivity and Specificity
  • Silver Staining / methods*

Substances

  • Fungal Proteins
  • Peptides
  • Proteins