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1.  Sulfonium Ion Derivatization, Isobaric Stable Isotope Labeling and Data Dependent CID- and ETD-MS/MS for Enhanced Phosphopeptide Quantitation, Identification and Phosphorylation Site Characterization 
An amine specific peptide derivatization strategy involving the use of novel isobaric stable isotope encoded ‘fixed charge’ sulfonium ion reagents, coupled with an analysis strategy employing capillary HPLC, ESI-MS, and automated data dependent ion trap CID-MS/MS, -MS3, and/or ETD-MS/MS, has been developed for the improved quantitative analysis of protein phosphorylation, and for identification and characterization of their site(s) of modification. Derivatization of 50 synthetic phosphopeptides with S,S′-dimethylthiobutanoylhydroxysuccinimide ester iodide (DMBNHS), followed by analysis using capillary HPLC-ESI-MS, yielded an average 2.5-fold increase in ionization efficiencies and a significant increase in the presence and/or abundance of higher charge state precursor ions compared to the non-derivatized phosphopeptides. Notably, 44% of the phosphopeptides (22 of 50) in their underivatized states yielded precursor ions whose maximum charge states corresponded to +2, while only 8% (4 of 50) remained at this maximum charge state following DMBNHS derivatization. Quantitative analysis was achieved by measuring the abundances of the diagnostic product ions corresponding to the neutral losses of ‘light’ (S(CH3)2) and ‘heavy’ (S(CD3)2) dimethylsulfide exclusively formed upon CID-MS/MS of isobaric stable isotope labeled forms of the DMBNHS derivatized phosphopeptides. Under these conditions, the phosphate group stayed intact. Access for a greater number of peptides to provide enhanced phosphopeptide sequence identification and phosphorylation site characterization was achieved via automated data-dependent CID-MS3 or ETD-MS/MS analysis due to the formation of the higher charge state precursor ions. Importantly, improved sequence coverage was observed using ETD-MS/MS following introduction of the sulfonium ion fixed charge, but with no detrimental effects on ETD fragmentation efficiency.
PMCID: PMC4228788  PMID: 21952753
Phosphopeptide fragmentation; Collision induced dissociation (CID); Electron transfer dissociation (ETD); Fixed charge
2.  Aberrant expression of CD133 protein correlates with Ki-67 expression and is a prognostic marker in gastric adenocarcinoma 
BMC Cancer  2010;10:218.
The relationships between the expression of CD133, Ki-67 and prognosis in gastric adenocarcinoma are unknown and needs exploring.
The samples of gastric adenocarcinoma from 336 Chinese patients with follow-up were analyzed for CD133 and Ki-67 protein expressions by immunohistochemical method.
CD133 was expressed in up to 57.4% (193/336) of this group of gastric carcinoma. The expression of CD133 was significantly higher in carcinoma than in normal (P = 0.0001) and dysplastic mucosas (P = 0.004). CD133 was positive corresponded with the tumour size, grade, infiltrative depth and clinical stage (all P < 0.05). The overall mean survival time of the patients with CD133 positive expression was shorter than that of patients with negative expression (P = 0.0001). The expression of CD133 has a positive correlation with that of Ki-67 (r = 0.188, P = 0.001) in gastric adenocarcinoma. CD133 was an independent prognostic indicator. (P = 0.0001).
It is suggested that CD133 may play an important role in the evolution of gastric adenocarcinoma and should be considered as a potential marker for the prognosis.
PMCID: PMC2891633  PMID: 20487522
3.  Aberrant Expression of ID2 protein and its correlation with EBV-LMP1 and P16(INK4A) in Classical Hodgkin Lymphoma in China 
BMC Cancer  2008;8:379.
The relationships between the expression of ID2, EBV-LMP1 and P16(INK4A) in Chinese classical Hodgkin lymphoma are unknown and need exploring.
Samples of classical Hodgkin lymphoma from 60 Chinese patients were analyzed for the expression of ID2, EBV-LMP1 and p16(INK4A) proteins by immunohistochemistry.
ID2 protein was expressed in 83.3% of this group of classical Hodgkin lymphoma, staining strongly in both cytoplasm and nucleus of the Hodgkin and Reed-Sternberg (HRS) cells. EBV-LMP1 and P16(INK4A) were overexpressed in 85.0% and 71.7% of Hodgkin lymphoma, respectively. EBV-LMP1 was noted in the cytoplasm, membrane and nucleus of HRS cells; P16(INK4A) was in the nucleus and cytoplasm. Microscopically, ID2, EBV-LMP1 and P16(INK4A) staining distinguished the HRS cells from the complex background of lymphocytes. ID2 was positively correlated with EBV-LMP1(P < 0.01), but P16(INK4A) was inversely related to EBV-LMP1 (P < 0.05).
It is suggested that ID2, EBV-LMP1 and P16(INK4A) could play an important role in the evolution of classical Hodgkin lymphoma, and be considered as potential adjunct markers to identify HRS cells in diagnosis.
PMCID: PMC2625365  PMID: 19099554

Results 1-3 (3)