MCF Protein Identification Flow Chart

					                                                                           Figure I

                                                 EMF Protein Analysis Flow Chart
                                                                   Biological material
                                                                   (Tissue, Cells, Body fluids)
                   HPLC/
                                                                                  Gel electrophoresis
                   Purification



              Isolated                                                        Gel                                                            Blotted on
              Protein                                                     protein spot                                                        PVDF
                                                                           (1D / 2D)                                                         membrane
                          Digestion/                                              Digestion/                                             Digestion/
                          cleavage                                                cleavage                                               cleavage




  Edman                                                                                                                                                     Edman
                 Amino Acid
degradation
                  Analysis
                                                                             Peptide mixture                                                              degradation
(sequence)                                                                                                                                                (sequence)
                 1 - 10 pmol
 500 fmol                                                                                                                       Desalting (ZipTip)        500 fmol
                                        HPLC fractionation                                 Proposed                              or Nanodesalting
                                                                                           Addition

                                  Fractionated                                                          MALDI-TOF          LC-ESI-MS         Nano ESI-MS
                                    peptide                                                             (mass map)         (mass map)         (mass map)
                                                                            LC-ESI-                       50 fmol          1 - 5 pmol        0.5 - 1 pmol
                                                                            MS/MS
                                                                           (MS scans plus
                                                                           MS/MS scans)
                                                                            mass map +
              PSD                   Nano               Edman                sequence tag                       PSD                               Nano
         MALDI-TOF              ESI-MS/MS            degradation        Q-Tof Ultima                      MALDI-TOF                           ESI-MS/MS
         (sequence tag)        (sequence tag)        (sequence)            50 - 500 fmol                  (sequence tag)                     (sequence tag)
            100 fmol            0.5 -1 pmol          300 fmol                                              100 fmol                        0.5 - 1 pmol




                                                               Database searches
                                                 (SEQUEST, Mascot, PROWL, Protein Prospector, etc.)

				
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