Secretome　(secreted　proteins)　has　been　arousing　considerable　attention　because　of　its　potential　to　develop　novel　biomarkers　for　the　early　detection　and　diagnosis　of　diseases.　Like　other　proteome　systems,　a　secretome　generally　consists　of　thousands　of　proteins　with　a　wide　dynamic　range　of　abundance.　The　effective　way　to　decrease　the　complexity　of　the　analyte　is　to　combine　different　prefractionation　methods　with　orthogonal　separation　capabilities.　The　newly　appeared　OFFGEL　fractionation　system　provides　the　ability　to　separate　the　peptides　into　discrete　liquid　fractions　according　to　their　differences　in　isoelectric　point　(pI),　and　has　demonstrated　high　efficiency　and　repeatability　for　the　analysis　of　the　complex　proteins.　Here　the　OFFGEL　fractionation　system　was　evaluated　for　the　separation　of　peptides　digests　from　secreted　proteins　of　hepatocellular　carcinoma　cells　(HCC),　using　a　12-wells　device　encompassing　the　pH　range　of　3　-　10.　In　combination　with　the　online　reversed　phase　liquid　chromatography　and　mass　spectrometry,　peptides　from　the　digestion　of　MHCC97L　secreted　proteins　were　separated　and　identified.　Preliminary　results　showed　that　nearly　80%　of　the　peptides　identified　were　specific　to　one　fraction,　and　40%　of　the　proteins　were　obtained　from　the　fractions　1　-3　from　the　acid　end.　The　average　of　peptide　pI　values　distributed　in　each　fraction　is　consistent　with　the　theoretical　pIs,　and　the　pI　distribution　of　the　peptides　focused　on　the　low　pH　range　showed　a　smaller　deviation.　Finally,　2　995　proteins　were　identified　from　secretome　of　MHCC97L　cell　lines.　Thus　the　data　show　that　the　OFFGEL　system　provides　a　highly　valuable　tool　to　fractionate　peptides　from　complex　protein　samples.