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).
Methods
Protein Mixture: A 50 protein mixture (5 pmol of
each protein) was digested using trypsin. 5 uL of
the digest supernatent (500 fmol per protein) was
loaded onto a 300 um i.d. x 15 cm C18 column (LC
Packings) and the peptides were separated using a
linear acetonitrile/water/TFA gradient (~5 L/min).
The LC eluent was automatically deposited onto an
unmodifi ed stainless steel MALDI target using the
AccuSpot MALDI spotting robot (Shimadzu, Japan).
Fractions were collected every 6 sec from T = 3
- 41 min. MALDI matrix solution (CHCA; 5 mg/mL in
50/50 acetonitrile/0.1% TFA) was added and mixed
with the LC eluent using the AccuSpot.
iTRAQ sample: 2 samples of an equimolar 5-protein mixture were labelled using the 114 and 117 reagents using the manufacturers recommended procedure. The two samples were combined and prepared for LCMALDI analysis using an SCX trap. 5 L of the digest mixture (500 fmol per protein) was loaded onto a C18 column and prepared as described above.
MALDI-MS and MALDI-MS/MS acquisitions were performed on an AXIMA-TOF2 mass spectrometer (Shimadzu Biotech/Kratos, UK). All acquisitions were performed in fully automated mode using the LC-MALDI software functionality of the MALDI-MS software. The minimum precursor intensity threshold was set to 30 mV and there was no limit on the number of candidates selected for MS/MS (i.e. all precursors meeting the acceptance criteria were selected). For MS/MS acquisitions, helium was used as the collision gas.
Results
Discussion
Protein Mixture Sample
Using the LC-MALDI technique described, we successfully identifi ed 33 p
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Source:
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