We present an improved method for MALDI-MS analysis
of proteins that have been electroblotted onto a nitrocellulose (NC) membrane. With this approach, electroblotted
proteins can be analyzed directly for intact molecular
weight determination or after on-membrane digestion by
dissolution of the nitrocellulose in MALDI matrix solution
containing 70% acetonitrile and 30% methanol. This
solution helps maintain solubility of proteins and peptides
while dissolving the NC membrane, which is dissolved
by 100% acetone in other protocols. On-membrane tryptic
digestion using this method requires half the time of ingel digestion and results in fewer missed cleavages and
better protein coverage. For the membrane proteins
studied, bovine uroplakins II and III, the protein coverage
was almost twice that provided by conventional in-gel
digestion, and the transmembrane domains of both
uroplakins were detected only after on-membrane digestion. We also demonstrated the compatibility with MALDIMS of a new dye, MemCode, which is specifically designed
for staining NC membrane-immobilized proteins and is
faster and more sensitive than Ponceau-S. Our improved
on-membrane digestion protocol greatly improves the
study of soluble and, particularly strikingly, integral
membrane proteins by mass spectrometry http://webdoc.nyumc.org/nyumc/files/sun-lab/attachments/2006.AC.MSa.pdf
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