Electrospray Ionization Mass Spectrometry of Oligonucleotides

Colette M. Castleberry1, Lenore P. Rodicio2, Patrick A. Limbach1

1 Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, University of Cincinnati, Cincinnati, Ohio, 2 Department of Natural and Social Sciences, Miami Dade College, InterAmerican Campus, Miami, Florida
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 10.2
DOI:  10.1002/0471142700.nc1002s35
Online Posting Date:  December, 2008
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Abstract

Because of the high molecular weights and thermal lability of biomolecules such as nucleic acids and protein, they can be difficult to analyze by mass spectrometry. Such analyses require a “soft” ionization method that is capable of generating intact molecular ions. In addition, most mass analyzers have a limited upper mass range that is not sufficient for studying these large molecules. ESI‐MS can be used to analyze molecules with a molecular weight that is larger than the mass‐to‐charge ratio limit of the analyzer. This unit describes how ESI allows for analysis of high‐molecular‐weight compounds through the generation of multiply charged ions in the gas phase. It discusses analyzer configurations and solvent selection, and gives protocols for sample preparation. For applications of ESI‐MS, the unit discusses molecular weight determination, sequencing, and analysis of oligonucleotide mixtures by LC‐MS. Curr. Protoc. Nucleic Acid Chem. 35:10.2.1‐10.2.19. © 2008 by John Wiley & Sons, Inc.

Keywords: Electrospray; mass spectrometry; ESI‐MS; nucleic acids; LC‐MS

     
 
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Table of Contents

  • Introduction
  • ESI‐MS Versus MALDI‐MS for Oligonucleotide Analysis
  • Instrument Configuration
  • Sample Preparation
  • Basic Protocol 1: Reduction of Salt Adduction in Oligonucleotide or Nucleic Acid Samples Using Ethanol Precipitation
  • Alternate Protocol 1: Preparation of Oligonucleotide or Nucleic Acid Samples for ESI‐MS with Chelating Agents and/or Organic Bases
  • Molecular Weight Determination of Oligonucleotides and Nucleic Acids
  • Sequencing Using ESI‐MS
  • Analysis of Noncovalent Complexes
  • LC‐MS of Oligonucleotides
  • Basic Protocol 2: Ion‐Pairing RP‐HPLC‐MS Analysis of Oligonucleotides Using TEAA Buffers
  • Support Protocol 1: Preparation of 2.0 M Triethylammonium Acetate
  • Alternate Protocol 2: Ion‐Pairing HPLC‐MS Analysis of Oligonucleotides Using HFIP Buffers
  • Support Protocol 2: Preparation of 800 mM 1,1,1,3,3,3‐Hexafluoroisopropanol (HFIP)
  • Basic Protocol 3: Anion‐Exchange HPLC‐MS Analysis of Oligonucleotides Using TEAA Buffers
  • Summary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Reduction of Salt Adduction in Oligonucleotide or Nucleic Acid Samples Using Ethanol Precipitation

  Materials
  • Oligonucleotide or nucleic acid of interest
  • 10 M ammonium acetate
  • Absolute ethanol, ice cold
  • 70% aqueous ethanol, ice cold

Alternate Protocol 1: Preparation of Oligonucleotide or Nucleic Acid Samples for ESI‐MS with Chelating Agents and/or Organic Bases

  Materials
  • Oligonucleotide or nucleic acid of interest
  • Appropriate organic solvent (see below): 30% to 70% (v/v) aqueous acetonitrile, isopropanol, or methanol
  • 0.1% (v/v) aqueous triethylamine (diluted from 99%, from Fisher)
  • trans‐1,2‐diaminocyclohexane‐N,N,N′,N′‐tetracetic acid (CDTA, molecular‐biology‐grade) or ethylenediaminetetraacetic acid (EDTA, molecular biology grade)

Basic Protocol 2: Ion‐Pairing RP‐HPLC‐MS Analysis of Oligonucleotides Using TEAA Buffers

  Materials
  • 2 M triethylammonium acetate (TEAA) buffer (see protocol 4)
  • H 2O, MS grade
  • 90% (v/v) MS‐grade acetonitrile in MS‐grade H 2O
  • Oligonucleotides of interest
  • Reversed‐phase C18 stationary phase column (recommended columns are Zorbax Eclipse XDB C18 from Agilent and Xterra MS C18 from Waters)

Support Protocol 1: Preparation of 2.0 M Triethylammonium Acetate

  Materials
  • Acetic acid (ACS reagent grade)
  • Triethylamine (99% pure, from Fisher)
  • HPLC‐grade water
  • 500‐mL funnel with stopcock or 50‐ or 100‐mL buret
  • Ring stand with ring to hold funnel or buret holder for buret
  • Magnetic stir plate with large stir bar
  • 250‐mL, 500‐mL, and 2‐L beakers
  • Graduated cylinders
  • Calibrated pH meter
  • 1‐L volumetric flask
NOTE: This is an exothermic reaction and should be carried out in an ice bath in the fume hood.

Alternate Protocol 2: Ion‐Pairing HPLC‐MS Analysis of Oligonucleotides Using HFIP Buffers

  Materials
  • 800 mM 1,1,1,3,3,3‐hexafluoroisopropanol (HFIP; see protocol 6)
  • H 2O, MS grade
  • Methanol, MS grade
  • Oligonucleotides of interest
  • Bath sonicator
  • Reversed‐phase C18 stationary phase column (recommended columns are Zorbax Eclipse XDB C18 from Agilent and Xterra MS C18 from Waters)

Support Protocol 2: Preparation of 800 mM 1,1,1,3,3,3‐Hexafluoroisopropanol (HFIP)

  Materials
  • 1,1,13,3,3‐Hexafluoroisopropanol (HFIP; 99% pure, Sigma‐Aldrich)
  • H 2O, HPLC grade
  • Triethylamine (99% pure, Fisher)
  • 100‐mL graduated cylinder
  • 2‐L beaker
  • Magnetic stir plate with large stir bar
  • 10 ml buret or sterile dropper
  • Calibrated pH meter

Basic Protocol 3: Anion‐Exchange HPLC‐MS Analysis of Oligonucleotides Using TEAA Buffers

  Materials
  • 2 M TEAA stock solution (see protocol 4)
  • 40% (v/v) acetonitrile (MS grade) in MS‐grade H 2O
  • MS‐grade water
  • MS‐grade acetonitrile
  • Anion‐exchange column (recommended column is ET 125/4 Nucleogen DEAE 60‐7)
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Figures

Videos

Literature Cited

Literature Cited
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