Biomedical Engineering Reference
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ion charge state will undoubtedly prove extremely useful in top-
down analyses.
Although methods for top-down proteomics continue to be
developed, comparatively few studies feature analysis of large
intact proteins, partly due to difficulties in gaining adequate pre-
cursor and product ion intensity from large, thermally labile ana-
lytes with polydisperse signals. Very few reports exist in the liter-
ature for proteins larger than 60 kDa, although promising recent
work with heated inlets (termed prefolding dissociation) indicates
4. Notes
1. Urea (up to 2 M) can be included in the ion exchange
mobile phases to enable dissociation of protein complexes
if this is desirable. If this is the case, ensure that samples are
not heated prior to the reversed-phase separation step as this
could result in irreversible protein carbamylation.
2. An advantage of using two ion exchange columns (a strong
anion exchanger and weak cation exchanger) in tandem is
that highly acidic proteins which would not be retained
on anion exchange material will interact with the cation
exchanger, and a common mobile phase will effect protein
elution from both columns.
3. At this stage a portion of the sample (10% should suffice) can
readily be removed for conventional reduction, alkylation,
proteolysis and LC-MS/MS analysis for additional identifi-
cation confirmation purposes.
Acknowledgements
I would like to thank current and former members of the Michael
Barber Centre, University of Manchester for constructive com-
ments during this work. John Cottrell (Matrix Sciences) pro-
vided demonstration licensing of top-down Mascot. Ken Cook
(Dionex) provided demonstration of HPLC columns and assisted
in setting up gradients. Carsten Baessmann, Andrea Kiehne,
Markus Lubeck, Andrea Schneider and Julia Smith (Bruker Dal-
tonics) have provided essential guidance both with ETD/PTR
experiments and with data processing.
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