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Detection of Disulfide-Linked Peptides by Mass Spectrometry

  • Alastair Aitken
  • Michèle Learmonth
Protocol
  • 97 Downloads
Part of the Springer Protocols Handbooks book series (SPH)

Abstract

Mass spectrometry is playing a rapidly increasing role in protein chemistry and sequencing (see  Chapters 91 and  97 100) and is particularly useful in determining sites of co- and posttranslational modification (1,2), and application in locating disulfide bonds is no exception. This technique can of course readily analyze peptide mixtures; therefore it is not always necessary to isolate the constituent peptides. However, a cleanup step to remove interfering compounds such as salt and detergent may be necessary. Thus can be achieved using matrices such as 10-μm porous resins slurry-packed into columns 0.25 mm diameter. Polypeptides can be separated on stepwise gradients of 5-75% acetonitrile in 0.1% formic or acetic acid (3). On-line electrospray mass spectrometry (ES-MS) coupled to capillary electrophoresis or high-performance liquid chromatography (HPLC) has proved particularly valuable in the identification of modified peptides. If HPLC separation on conventional columns is attempted on-line with mass spectrometry, the level of trifluoroacetic acid (TFA) (0.1 %) required to produce sharp peaks and good resolution of peptides results in almost or complete suppression of signal. In this case it is recommended to use the new “low TFA,” 218MS54, reverse-phase HPLC columns from Vydac (300 Å pore size) which can be used with as little as 0.005% TFA without major loss of resolution and minimal signal loss (see further details in  Chapter 85).

Keywords

Disulfide Bond Cyanogen Bromide Modify Peptide Intramolecular Disulfide Bond Performic Acid 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Humana Press Inc., Totowa, NJ 2002

Authors and Affiliations

  • Alastair Aitken
    • 1
  • Michèle Learmonth
    • 2
  1. 1.Division of Biomedical and Clinical Laboratory Sciences, Membrane Biology GroupUniversity of EdinburghScotland, UK
  2. 2.Department of Biomedical SciencesUniversity of EdinburghScotland

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