Proteomic analysis of the human colon carcinoma cell line (LIM 1215): Development of a membrane protein database

The proteomic definition of plasma membrane proteins is an important initia l step in searching for novel tumor marker proteins expressed during the di fferent stages of cancer progression. However, due to the charge heterogene ity and poor solubility of membrane-associated proteins this subsection of the cell's proteome is often refractory to two-dimensional electrophoresis (2-DE), the current paradigm technology for studying protein expression pro files. Here, we describe a non-2-DE method for identifying membrane protein s. Proteins from an enriched membrane preparation of the human colorectal c arcinoma cell line LIM1215 were initially fractionated by sodium dodecyl su lfate-polyacrylamide gel electrophoresis (SDS-PAGE, 4-20%). The unstained g el was cut into 16 x 3 mm slices, and peptide mixtures resulting from in-ge l tryptic digestion of each slice were individually subjected to capillary- column reversed phase-high performance liquid chromatography (RP-HPLC) coup led with electrospray ionization-ion trap- mass spectrometry (ESI-IT-MS). I nterrogation of genomic databases with the resulting collision-induced diss ociation (CID) generated peptide ion fragment data was used to identify the proteins in each gel slice. Over 284 proteins (including 92 membrane prote ins) were identified, including many integral membrane proteins not previou sly identified by 2-DE, many proteins seen at the genomic level only, as we ll as several proteins identified by expressed sequence tags (ESTs) only. A dditionally, a number of peptides, identified by de novo MS sequence analys is, have not been described in the databases. Further, a "targeted" ion app roach was used to unambiguously identify known low-abundance plasma membran e proteins, using the membrane-associated A33 antigen, a gastrointestinal-s pecific epithelial cell protein, as an example. Following localization of t he A33 antigen in the gel by immunoblotting, ions corresponding to the theo retical A33 antigen tryptic peptide masses were selected using an "inclusio n" mass list for automated sequence analysis. Six peptides corresponding to the A33 antigen, present at levels well below those accessible using the s tandard automated "nontargeted" approach, were identified. The membrane pro tein database may be accessed via the World Wide Web (WWW) at http://www.lu dwig.edu.au/jpsl/jpslhome.html.