Transcript MCF Protein Identification Flow Chart

Figure I
EMF Protein Analysis Flow Chart
Biological material
(Tissue, Cells, Body fluids)
HPLC/
Purification
Gel electrophoresis
Digestion/
cleavage
Digestion/
cleavage
Edman
degradation
(sequence)
 500 fmol
Amino Acid
Analysis
1 - 10 pmol
Digestion/
cleavage
Peptide mixture
Fractionated
peptide
Nano
ESI-MS/MS
(sequence tag)
0.5 -1 pmol
LC-ESIMS/MS
Edman
degradation
(sequence)
 300 fmol
Desalting (ZipTip)
or Nanodesalting
Proposed
Addition
HPLC fractionation
PSD
MALDI-TOF
(sequence tag)
 100 fmol
Blotted on
PVDF
membrane
Gel
protein spot
(1D / 2D)
Isolated
Protein
(MS scans plus
MS/MS scans)
mass map +
sequence tag
Q-Tof Ultima
50 - 500 fmol
MALDI-TOF
(mass map)
 50 fmol
PSD
MALDI-TOF
(sequence tag)
 100 fmol
Database searches
(SEQUEST, Mascot, PROWL, Protein Prospector, etc.)
LC-ESI-MS
(mass map)
1 - 5 pmol
Edman
degradation
(sequence)
 500 fmol
Nano ESI-MS
(mass map)
0.5 - 1 pmol
Nano
ESI-MS/MS
(sequence tag)
0.5 - 1 pmol