Glycoprotein is a kind of proteins widely distributed in the bodies of animals and plants. The identification of its characteristic is very difficult due to the complexity of its structure. In this paper, a serious of methods for glycoprotein analysis was established by mass spectrum. The molecular weight, quantity of N-glycans and sialic acids were determined by matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOFMS) directly. The glycosylation sites of glycoprotein were confirmed by its PMF,which produced by the combination digestion of PNGase F and Endoproteinase. The sequence of glycopeptide was detected by liquid chromatography-mass spectrometry (LCMS). The recombined biotechnologic productions including rhEPO,proUK and Spike protein of SARS coronavirus were well characterized by using these methods.
In proteomics study, because there were biases of two-dimensional gel electrophoresis in molecular weights, pH and hydrophobicity, multi-dimensional protein identification technology (MuD-PIT)was proposed. Due to the core part of the technique being digestion of total proteins in the first step, some drawbacks appeared, such as complicating the separation of samples, dominating the influence of proteins with high abundance on the identification of proteins with low abundance and losing some information of proteins,for example, post-translational modification et al. , these resulted in the limitation of applications for the method. Taking into account of those factors mentioned above, when studying the mitochondria proteome of human fetal liver with great significance in life process, Proteome Lab PF2D system (Beckman Coulter, USA) was employed. The fractions of extract from fetal liver tissues by saccharose- density gradient centrifugation were prepared on a high velocity centrifuge. The desalting and solvent exchanging of the fractions with Start buffer (pH8. 5, Beckman Coulter, USA) were carried out on a PD-10(Sephadex G-25, 5 000Da) desalting column (amersham pharmacia biotech, Wikstroms,Sweden) according to the procedures described in its product instruction. Then the treated fractions were separated with chromatofocusing and reversed-phase liquid chromatography in tandem, respectively. After concentration and in-solution digestion of protein fractions,the peptide mixture was lyophilized, then resolved with 5% aqueous acytonitrile plus 0.1% formic acid, and finally analyzed with microLC-ESI-ion trap mass spectrometry(Thermo Finnigan, San Jose, CA, USA) and CapLC-ESI-QTOF mass spectrometry (Waters, USA). Mass spectrometric data was searched against IPI protein database with Thermo SEQUEST (Thermo Finnigan, San Jose, CA, USA) and Mascot. A primary database of mitochondrial proteome of human fetal liver was constructed, which could play central role in the large-scale study of the functions of human liver proteo
