| Glycosylation is one of the most common protein post-translationalmodifications. More than50%of human proteins have been considered to containglycosylation. Glycosylation of proteins plays an important role in the variousbiological processes, such as immune response, reproductive process, neuraldevelopment, apoptosis and senescence, tumor development and metastasis, et. al. Itis well established that altered glycosylation varies significantly in the tumordevelopment. It has been reported that typical changes include increased levels offucose, sialic acid, the addition of polylactosamine units (repeating sequences ofgalactose and Nacetylglucosamine), and higher-ordered branching of N-linkedglycans. Although the functions of these specific type of glycans aberrant intumorigenesis and the mechanism is not very clear, the use of type-specific changes inthese glycans as potential biomarkers has become possible.As a traditional indicator for HCC（hepatocellular carcinoma）testing, diagnosticvalue of AFP（alpha fetoprotein, AFP）has been evaluated and questioned. In fact, theconcentration of AFP sometimes elevated in the germ cell tumors (nonseminomatousgerm cell tumor，NSGCT), intrahepatic cholangiocarcinoma (non-seminomatous,ICC) and gastric carcinoma patients. The proportion of chronic hepatitis patients withevaluated concentration of serum AFP is15%to58%, which is11%to47%in livercirrhosis patients. These cases will lead to decreasing specificity of AFP testing.Especially in patients with chronic liver disease at high risk of developing HCC,diagnostic efficiency of AFP testing will be significantly impaired. Previous reportssuggest that AFP heterogeneity with various glycans can be used as an independentbiomarker in detecting hepatocellular carcinoma. AFP-L3, the main component ofserum AFP in HCC patients has been approved as a HCC biomarker by the FDA. Ithas been reported that the detection sensitivity of AFP-L3assay and AFP assay aresimilar, but the former specificity was92.5%and the latter was75.3%.Currently, the major methods to detect AFP glycol-isoforms are based onspecific affinity of different lectins to various oligosaccharide chains. These detectionmethods have their limitations. First, the specific lectin can only affinity to the certaintype of glycan, therefore, it may not be possible to distinguish between differentoligosaccharides with the same glycans recognized by one type of lectin. Secondly,lectin-based detection reflects the changes in all type of oligosaccharides instead ofthe changes at certain glycosylation site. To solve these problems, we propose acombination strategy with immunoaffinity enrichment and multiple reactionmonitoring (MRM) technology to quantitatively analyze various oligosaccharides of serum AFP.In the first part of this paper, using the state-of-the-art mass spectrometer withhigh resolution and high accuracy, we have established a method to qualitativelyanalyze the intact and simplified glycopeptides. Compared with the traditionalanalytical strategies based on MALDI-TOF mass spectrometer, our method has thefollowing advantages: First, solving loss of information at specific glycosylation siteresulting from separation oligosaccharides from peptides in traditional method.Secondly, the required sample amount in our assay is much lower than that ofconventional method. Only loading500fmol standard protein sample, a wealth ofinformation of glycopeptides can be obtained to determine the oligosaccharidesstructure. Thirdly, our technology facilitates the detection of sialylated glycopeptides,which are difficult to be identified by conventional methods partly due to frangibilityof sialic acid in MALDI-TOF mass spectrometer.Finally, we identified16AFP glycopeptides which can be used for thedevelopment MRM based glycopeptide analytical method in the next study..In the second part of this paper, we have established a MRM based analyticalmethod forAFP glycopeptides detection. We systematically explored fragmentationbehavior of the intact glycopeptides and a simplified glycopeptide in the triplequadrupole mass spectrometer, optimized the collision energy ofglycopeptidesfragmentation, compared the performance of quantitative analysis among differentoligosaccharides residues, first proposed that oligosaccharides residues m/z138and366are optimal daughter ions for identifying glycopeptides and the quantitativeresults with these two ions are highly consistent. Then we systematically evaluateddetection limit, linear range, reproducibility of the method. Finally, using optimizedMRM method, we successfully performed a quantitative analysis of the intact andsimplified glycopeptides of cord blood AFP. Compared with the MRM methodpreviously developed by our labarotatory to quantitatively analyze the simplified corefucosylation glycopeptides, the establishment of the method in this study pushedforward this research to a higher and intact glycopeptides level. To more accuratelyreflect the AFP changes,we utilized the ratio of the intact or simplified glycopeptidesto a endogenous AFP peptide as a indictor, taking into account both the change inglycosylation and that in the total protein amount..In the third part of this paper, we developed a strategy which combined theimmunoaffinity enrichment with the mass spectrometric quantitative analysis. Inactual serum samples, detection limit of the strategy is10ng/mL. Using optimizedaffinityimmune-MRM method, we explored the changes of16intact glycopeptidesand a simplified glycopeptide of AFP treated with Endo F3endoglycosidase in thegroup of15liver cirrhosis patients and that of22HCC patients. The results suggestedthat the ratio of glycopeptides G3, G6, G13and the simplified glycopeptide to a endogenous AFP peptide had significant difference between patients withhepatocellular carcinoma (HCC) and liver cirrhosis (LC). which mightbe a promisingresult for develop novel tumor marker for liver cancer.In the fourth part of this paper, we conducted a systematic study to explore thepeptides alkylation under the normally used protein digestion conditions,The impactof over alkylation on qualitative and quantitative analysis of proteome was evaluated.In the last part of this paper, we have used stable isotope labeling by amino acidsin cell culture (SILAC) to determine quantitative differences in host proteins afterinfection of human lung A549cells with highly pathogenic avian influenza H5N1for24h. Of the3504proteins,72were significantly up-regulated,66were significantlydown-regulated. Gene ontology and pathway analyses were performed to annotate thedifferentially regulated proteins, and the result indicated some of these differentiallyexpressed proteins involved in host cell molecular regulation and signal transductionpathways. The study provides a very meaningful clue for the study of pathogenicmolecular mechanism of highly pathogenic avian influenza H5N1. |
