Strategic shotgun proteomics approach for efficient construction of an expression map of targeted protein families in hepatoma cell lines

Abstract

An expression map of the most abundant proteins in human hepatoma HepG2 cells was established by a combination of complementary shotgun proteomics approaches. Two-dimensional liquid chromatography (LC)-nano electrospray ionization (ESI) tandem mass spectrometry (MS/MS) as well as one-dimensional LC-matrix-assisted laser desorption/ionization MS/MS were evaluated and shown that additional separation introduced at the peptide level was not as efficient as simple prefractionation of protein extracts in extending the range and total number of proteins identified. Direct LCnanoESI MS/MS analyses of peptides from total solubilized fraction and the excised gel bands from one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis fractionated insolubilized fraction afforded the best combination in efficient construction of a nonredundant cell map. Compiling data from multiple variations of rapid shotgun proteomics analyses is nonetheless useful to increase sequence coverage and confidence of hits especially for those proteins identified primarily by a single or two peptide matches. While the returned hit score in general reflects the abundance of the respective proteins, it is not a reliable index for differential expression. Using another closely related hepatoma Hep3B as a comparative basis, 16 proteins with more than two-fold difference in expression level as defined by spot intensity in two-dimensional gel electrophoresis analysis were identified which notably include members of the heat shock protein (Hsp) and heterogeneous nuclear ribonucleoprotein (hnRPN) families. The observed higher expression level of hnRNP A2/B1 and Hsp90 in Hep3B led to a search for reported functional roles mediated in concert by both these multifunctional cellular chaperones. In agreement with the proposed model for telomerase and telomere bound proteins in promoting their interactions, data was obtained which demonstrated that the expression proteomics data could be correlated with longer telomeric length in tumorigenic Hep3B. This biological significance constitutes the basis for further delineation of the dynamic interactions and modifications of the two protein families and demonstrated how proteomic and biological investigation could be mutually substantiated in a productive cycle of hypothesis and pattern driven research.

Enhanced nuclear factor-kappa B-associated Wnt-1 expression in hepatitis B- and C-related hepatocarcinogenesis: identification by functional proteomics

“…A modified silver staining method compatible with mass spectrometric analysis provided protein spot visualization was used for the study [27]. Briefly, gels were fixed in 50% methanol, 10% acetic acid in water for 30 min followed by incubation in 5% methanol for 15 min.…”

“…The theoretical Mr and pI values of the 2-DE markers were used to calibrate the Mr and pI of the protein spots on the 2-DE gels. Intensity levels were normalized between gels as a proportion of total protein intensity detected for the entire gel and the protein quantity of each spot was calculated by integrating density over the spot area [27,28].…”

“…Two-dimensional polyacrylamide gel electrophoresis (2-DE) was carried out according to standard protocols [26], and the detailed procedures as described previously [27].…”

“…Tryptic peptides from 2-DE protein spots were subjected to peptide mass fingerprinting (PMF) using a matrix-assisted laser desorption/ionization quadrupole-time of flight (MALDI-Q-TOF) mass spectrometer (M@LDI TM ; Micromass, Manchester, UK) operated in reflectron positive ion mode as described [27][28][29]. Briefly, samples were spotted onto the 96-well format MALDI target plate using a saturated matrix solution of a-cyano-4-hydroxycinnamic acid (CHCA) in 60% ACN/1% TFA.…”

Proteomics for the early detection and treatment of hepatocellular carcinoma

“…Expression maps of proteins expressed in hepatoma cell lines have been constructed (Lee et al, 2003; http:// proteome.btc.nus.edu.sg/hccm/). These maps will facilitate integration of studies performed in different groups worldwide.…”