Protein Protocols Protein Protocols

Disulfide-Linked Peptide Detection by MS 585
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Detection of Disulfide-Linked Peptides
by Mass Spectrometry
Alastair Aitken and Michèle Learmonth
1. Introduction
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).
Sequence information is readily obtained using triple quadrupole tandem mass
spectrometry after collision-induced disassocation (4). Ion trap mass spectrometry
technology (called LCQ) is now well established which also permits sequence
information to be readily obtained. Not only can MS-MS analysis be carried out, but
owing to the high efficiency of each stage, further fragmentation of selected ions may
be carried out to MS.” The charge state of peptide ions is readily determined by a
“zoom-scan” technique that resolves the isotopic envelopes of multiply charged peptide
ions. The instrument still allows accurate molecular mass determination to 100,000 Da
at 0.01% mass accuracy. The recent development of Fourier transform ion cyclotron
resonance mass spectrometry (5), in which the ions can be generated by a wide variety
of techniques, has very high resolution and sensitivity.
From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
585