To further identify these proteins, the gel bands at about 69 kDa, 53 kDa, 47 kDa, 38 kDa and 35 kDa were excised from the gel separately and subjected to in-gel digestion. Finally, these bands were analyzed using mass spectroscopy to determine appropriate information about the proteins. The obtained data were addressed using Mascot2.2 software. After searching the database on the Unified Protein Database (UniProt, for detailed information please see the supporting information), a series of related peptides were identified. Based on the sequence of these peptides and information gleaned from the database, the sequences of these proteins were determined. And the related VDPs were identified (Figure and Table ). In accordance with the results from live cell imaging, most of the identified VDPs are present in the mitochondria and (or) plasma membranes (Table , detailed mass spectroscopic information and amino acid sequence, as well as their cellular distribution for the proteins are listed in the supporting information). In particular, the thioredoxin reductase 1 (TrxR1), protein disulfide-isomerase (PDI), medium-chain specific acyl-CoA dehydrogenase and malate dehydrogenase (MDH) were identified. These results are consistent with previous results. Interestingly, the lane loaded with DTNB-treated cell lysis displays a decrease in the fluorescence signal for the 53 kDa and 38 kDa band, which indicated the presence of cell-surface VDPs for these protein bands. After searching the database UniProt, we deduced that the 53-kDa protein band may belong to the PDI family (Swiss-Prot accession number is B3KQT2 and B3KQT9), which was previously reported on the endoplasmic reticulum (ER). The 38-kDa protein band could be assigned to Annexin A2 which is a new type of VDP where the vicinal dithiols may be at residue Cys133 and Cys262. Annexin A2 is an important functional protein that has been proposed as a potential biomarker and therapeutic target for cancer and antivirus-drug discovery. Herein, our results confirmed that some PDIs and Annexin A2 are cell-membrane expressed VDPs in MCF-7 cells. While, the unique peptide sequences of the protein disulfide-isomerase A6 (PDIA6) and Alpha-enolase (ENO1) were also identified from the protein band at about 48 kDa using mass spectroscopy (Figure and Table ). Using sequence information from UniProt, we deduced that PDIA6 and ENO1 may also be expressed in MCF-7 cells. However, no fluorescence signal decrease was observed for the 48-kDa protein band in the lane with DTNB-treated cell lysis, suggesting that PDIA6 and ENO1 are expressed mainly in intracellular organelles and cytosol. However, we cannot confirm from just that observation that the PDIA6 and ENO1 do not localize on the cell membrane of MCF-7 cells, since PDIA6 and ENO1 have previously been detected on the surface of several cell types. Similarly, MDH at the 35-kDa protein band could also localize in the mitochondria and cell membrane. All the above results indicated that various functional VDPs were expressed in the MCF-7 cells. And these VDPs localized mainly in the mitochondria and cell membrane. Significantly, by inhibiting the cell membrane thiols with DTNB, our results confirmed the expression of some PDIs and Annexin A2 in the cell membrane of MCF-7 cells. Considering the important role of the PDIs family and Annexin A2 in cancer cells, we believe that our results will inspire the design of new drug candidates for targeting cell-surface PDIs or Annexin A2 in breast cancer therapy, as well as promoting the development of new diagnostic tools for cancer therapy.