Rapid and efficient glycoprotein identification through microwave-assisted enzymatic digestion

Identification of protein glycosylation sites is analytically challenging due to the diverse glycan structures associated with a glycoprotein. Mass spectrometry (MS)-based identification and characterization of glycoproteins has been achieved predominantly with the bottom-up approach, which typically involves the enzymatic cleavage of proteins to peptides prior to LC/MS or LC/MS/MS analysis. However, the process can be challenging due to the structural variations and steric hindrance imposed by the attached glycans. Alternatives to conventional heating protocols, that increase the rate of enzymatic cleavage of glycoproteins, may aid in addressing these challenges. An enzymatic digestion of a glycoprotein can be accelerated and made more efficient through microwave-assisted digestion. In this paper, a systematic study was conducted to explore the efficiency of microwave-assisted enzymatic (trypsin) digestion (MAED) of glycoproteins as compared with the conventional method. In addition, the optimum experimental parameters for the digestion such as temperature, reaction time, and microwave radiation power were investigated. It was determined that efficient tryptic digestion of glycoproteins was attained in 15 min, allowing comparable if not better sequence coverage through LC/MS/MS analysis. Optimum tryptic cleavage was achieved at 45°C irrespective of the size and complexity of the glycoprotein. Moreover, MAED allowed the detection and identification of more peptides and subsequently higher sequence coverage for all model glycoprotein. MAED also did not appear to prompt a loss or partial cleavage of the glycan moieties attached to the peptide backbones.     

A procedure for the analysis of site‐specific and structure‐specific fucosylation in alpha‐1‐antitrypsin

A MS‐based methodology has been developed for analysis of core‐fucosylated versus antennary‐fucosylated glycosites in glycoproteins. This procedure is applied to the glycoprotein alpha‐1‐antitrypsin (A1AT), which contains both core‐ and antennary‐fucosylated glycosites. The workflow involves digestion of intact glycoproteins into glycopeptides, followed by double digestion with sialidase and galactosidase. The resulting glycopeptides with truncated glycans were separated using an off‐line HILIC (hydrophilic interaction liquid chromatography) separation where multiple fractions were collected at various time intervals. The glycopeptides in each fraction were treated with PNGase F and then divided into halves. One half of the sample was applied for peptide identification while the other half was processed for glycan analysis by derivatizing with a meladrazine reagent followed by MS analysis. This procedure provided site‐specific identification of glycosylation sites and the ability to distinguish core fucosylation and antennary fucosylation via a double digestion and a mass profile scan. Both core and antennary fucosylation are shown to be present on various glycosites in A1AT.     

Identification of rat urinary glycoproteome captured by three lectins using gel and LC-based proteomics

Many different types of urine proteome studies have been done, but urine glycoprotein studies are insufficient. Therefore, we studied the glycoproteins from rat urine, which could be used to identify biomarkers in an animal model. First, urinary proteins were prepared by using the dialysis and lyophilizing methods from rat urine. Glycoproteins enriched with lectin affinity purification, concanavalin A, jacalin and wheat germ agglutinin from the urinary proteins were separated by means of reverse-phase fast protein LC (FPLC) or 1-D PAGE. Each FPLC fraction and 1-D PAGE gel band were trypsin-digested and analyzed by means of nanoLC-MS/MS. LC-MS/MS analyses were carried out by using linear ion trap MS. A total of 318 rat urinary glycoproteins were identified from the FPLC fractions and gel bands; approximately 90% of identified proteins were confirmed as glycoproteins in Swiss-Prot. Many glycoproteins, known as biomarkers, including C-reactive protein, uromodulin, amyloid beta A4 protein, alpha-1-inhibitor 3, vitamin D-binding protein, kallikrein 3 and fetuin-A were identified in this study. By studying urinary glycoproteins collected from rat, these results may help to assist in identifying urinary biomarkers regarding various types of disease models.     

Characterization of a gel-separated unknown glycoprotein by liquid chromatography/multistage tandem mass spectrometry: analysis of rat brain Thy-1 separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis

We developed an efficient and convenient strategy for protein identification and glycosylation analysis of a small amount of unknown glycoprotein in a biological sample. The procedure involves isolation of proteins by electrophoresis and mass spectrometric peptide/glycopeptide mapping by LC/ion trap mass spectrometer. For the complete glycosylation analysis, proteins were extracted in intact form from the gel, and proteinase-digested glycoproteins were then subjected to LC/multistage tandem MS (MSn) incorporating a full mass scan, in-source collision-induced dissociation (CID), and data-dependent MSn. The glycopeptides were localized in the peptide/glycopeptide map by using oxonium ions such as HexNAc+ and NeuAc+, generated by in-source CID, and neutral loss by CID-MS/MS. We conducted the search analysis for the glycopeptide identification using search parameters containing a possible glycosylation at the Asn residue with N-acetylglucosamine (203 Da). We were able to identify the glycopeptides resulting from predictable digestion with proteinase. The glycopeptides caused by irregular cleavages were not identified by the database search analysis, but their elution positions were localized using oxonium ions produced by in-source CID, and neutral loss by the data-dependent MSn. Then, all glycopeptides could be identified based on the product ion spectra which were sorted from data-dependent CID-MSn spectra acquired around localized positions. Using this strategy, we successfully elucidated site-specific glycosylation of Thy-1, glycosylphosphatidylinositol (GPI)-anchored proteins glycosylated at Asn23, 74, and 98, and at Cys111. High-mannose-type, complex-type, and hybrid-type oligosaccharides were all found to be attached to Asn23, 74 and 98, and four GPI structures could be characterized. Our method is simple, rapid and useful for the characterization of unknown glycoproteins in a complex mixture of proteins.     

Two-step protease digestion and glycopeptide capture approach for accurate glycosite identification and glycoprotein sequence coverage improvement

A novel two-step protease digestion and glycopeptide capture approach has been developed. It is different from traditional tryptic digestion, glycopeptide enriching and identification approach in glycoproteomics. Here, proteins were first digested by Lys-C into relatively large peptides. Glycopeptides among them were selectively captured by hydrazide resin through oxidized glycans. After thorough washing steps, trypsin was used as a second protease to in situ release non-glycosylated part (named as LT-peptides) from glycopeptides. Subsequently, the remaining part of glycopeptides on resin was de-glycosylated by peptide-N-glycosidase F, and collected as DG-peptides. Finally, both LT- and DG-peptides could be analyzed by mass spectrometer, achieving glycoprotein and glycosite identification. The approach was applied to cell lysate after positive validation by a model glycoprotein: 143 N-glycoproteins identified from DG- and LT-fraction both. In those glycoproteins, 189 DG-peptide-revealed N-glycosites got further confirmation by neighboring LT-peptides, which, in the meantime, made 109 glycoproteins get improved sequence coverage with increase even up to 350% (averagely 79.4%). Through controllable release, separate identification and combined interpretation of non-glycopeptides (newly introduced LT-peptides here) and traditional de-glycopeptides, the approach could not only achieve routine N-glycosite identification, but also provide further proofs of N-glycosites and increase glycoprotein sequence coverage.     

Site-specific N-glycosylation analysis: matrix-assisted laser desorption/ionization quadrupole-quadrupole time-of-flight tandem mass spectral signatures for recognition and identification of glycopeptides

The identification of glycosylation sites in proteins is often possible through a combination of proteolytic digestion, separation, mass spectrometry (MS) and tandem MS (MS/MS). Liquid chromatography (LC) in combination with MS/MS has been a reliable method for detecting glycopeptides in digestion mixtures, and for assigning glycosylation sites and glycopeptide sequences. Direct interfacing of LC with MS relies on electrospray ionization, which produces ions with two, three or four charges for most proteolytic peptides and glycopeptides. MS/MS spectra of such glycopeptide ions often lead to ambiguous interpretation if deconvolution to the singly charged level is not used. In contrast, the matrix-assisted laser desorption/ionization (MALDI) technique usually produces singly charged peptide and glycopeptide ions. These ions require an extended m/z range, as provided by the quadrupole-quadrupole time-of-flight (QqTOF) instrument used in these experiments, but the main advantages of studying singly charged ions are the simplicity and consistency of the MS/MS spectra. A first aim of the present study is to develop methods to recognize and use glycopeptide [M+H]+ ions as precursors for MS/MS, and thus for glycopeptide/glycoprotein identification as part of wider proteomics studies. Secondly, this article aims at demonstrating the usefulness of MALDI-MS/MS spectra of N-glycopeptides. Mixtures of diverse types of proteins, obtained commercially, were prepared and subjected to reduction, alkylation and tryptic digestion. Micro-column reversed-phase separation allowed deposition of several fractions on MALDI plates, followed by MS and MS/MS analysis of all peptides. Glycopeptide fractions were identified after MS by their specific m/z spacing patterns (162, 203, 291 u) between glycoforms, and then analyzed by MS/MS. In most cases, MS/MS spectra of [M+H]+ ions of glycopeptides featured peaks useful for determining sugar composition, peptide sequence, and thus probable glycosylation site. Peptide-related product ions could be used in database search procedures and allowed the identification of the glycoproteins.     

Analysis of intact erythropoietin and novel erythropoiesis-stimulating protein by capillary electrophoresis-electrospray-ion trap mass spectrometry

Recombinant human erythropoietin (rHuEPO) and novel erythropoiesis-stimulating protein (NESP) were analyzed by CE-ESI-MS using an IT as analyzer. The IT parameters were optimized by direct infusion of solutions of different intact proteins (myoglobin, transferrin, alpha1-acid glycoprotein and fetuin) with different degrees of glycosylation (from 0 to 35% w/w). Two physically adsorbed capillary coatings from UltraTol Pre-Coats (low normal (LN) and high reverse (HR)) were evaluated for the separation of rHuEPO and NESP glycoforms by CE-ESI-IT-MS. The results obtained with the neutral LN coating suggest that an IT mass spectrometer enables identification of the main glycoforms of a complex glycoprotein such as rHuEPO. Although LN provided acceptable glycoform resolution for rHuEPO, the separation obtained for NESP was less significant due to the higher microheterogeneity of this glycoprotein. Reproducibility studies confirmed the lack of stability and bleeding of the LN coating, which caused problems with MS detection, such as a dramatic loss of sensitivity and the presence of peaks in the mass spectra corresponding to molecular ions in the coating. In contrast, the cationic HR coating gave faster but poorer glycoform separations due to the presence of an anodal EOF. However, the positive charge of the coating provided enhanced hydrolytic stability, making it more suitable than the LN coating for the on-line MS coupling.     

水溶性硼酸亲和富集材料的制备及基于酶联免疫吸附法检测人肝微粒体糖蛋白的应用

生物样品中的糖蛋白丰度低,且在检测中易受到其它非糖蛋白的抑制和干扰,需在分析检测前对糖蛋白进行富集,但常规的基于固相材料的糖蛋白富集方法不易与生物技术中最经典的酶联免疫吸附法(ELISA)兼容.本研究以树枝状聚合物PAMAM 4.0为载体, 结合硼酸亲和技术,制备了新型水溶性硼酸亲和富集材料(DBC),并将其应用于基于ELISA的人肝微粒体中糖蛋白的检测.采用标准糖蛋白对DBC富集条件进行优化,然后考察其灵敏度和抗干扰能力,将优化后的方法应用于复杂样品人肝微粒体糖蛋白富集.结果表明,DBC对糖蛋白的富集选择性可高达100000倍,可将糖蛋白的富集信号提高100倍.以DBC为富集材料,与ELISA分析技术相结合,只需一步简单的孵育,即可实现生物样品中糖蛋白的高灵敏度、高选择性检测,为疾病相关的糖蛋白组学研究提供了一种有效的检测手段.     

Isotope tag method for quantitative analysis of carbohydrates by liquid chromatography-mass spectrometry

We have previously demonstrated that liquid chromatography/mass spectrometry equipped with a graphitized carbon column (GCC-LC/MS) is useful for the structural analysis of carbohydrates in a glycoprotein. Here, we studied the monosaccharide composition analysis and quantitative oligosaccharide profiling by GCC-LC/MS. Monosaccharides were labeled with 2-aminopyridine and then separated and monitored by GCC-LC/MS in the selective ion mode. The use of tetradeuterium-labeled pyridylamino (d4-PA) monosaccharides as internal standards, which were prepared by the tagging of standard monosaccharides with hexadeuterium-labeled 2-aminopyridine (d6-AP), afforded a good linearity and reproducibility in ESIMS analysis. This method was successfully applied to the monosaccharide composition analysis of model glycoproteins, fetuin, and erythropoietin. For quantitative oligosaccharide profiling, oligosaccharides released from an analyte and a standard glycoprotein were tagged with d0- and d6-AP, respectively, and an equal amount of d0- and d4-PA oligosaccharides were coinjected into GCC-LC/MS. In this procedure, the oligosaccharides that existed in either analyte or a standard glycoprotein appeared as single ions, and the oligosaccharides that existed in both analyte and a standard glycoprotein were detected as paired ions. The relative amount of analyte oligosaccharides could be determined on the basis of the analyte/internal standard ion-pair intensity ratio. The quantitative oligosaccharide profiling enabled us to make a quantitative and qualitative comparison of glycosylation between the analyte and standard glycoproteins. The isotope tag method can be applicable for quality control and comparability assessment of glycoprotein products as well as the analysis of glycan alteration in some diseases.     

Improved conditions for periodate/Schiff's base‐based fluorescent staining of glycoproteins with dansylhydrazine in SDS‐PAGE

An improved periodate/Schiff's base based fluorescent stain with dansylhydrazine (DH) for glycoproteins in 1D and 2D SDS‐PAGE was described. Down to 4–8 ng of glycoproteins can be selectively detected within 2 h, which is approximately 16‐fold higher than that of original protocol, but similar to that of Pro‐Q Emerald 488 stain (Invitrogen, Carlsbad, USA). Furthermore, subsequent study of deglycosylation, glycoprotein affinity isolation, and LC‐MS/MS analysis were performed to confirm the specificity of the improved method. As a result, improved DH stain may provide a new choice for selective, economic, MS compatible, and convenient visualization of gel‐separated glycoproteins.     

Comparison of HPLC/ESI-FTICR MS versus MALDI-TOF/TOF MS for glycopeptide analysis of a highly glycosylated HIV envelope glycoprotein

Defining the structures and locations of the glycans attached on secreted proteins and virus envelope proteins is important in understanding how glycosylation affects their biological properties. Glycopeptide mass spectrometry (MS)-based analysis is a very powerful, emerging approach to characterize glycoproteins, in which glycosylation sites and the corresponding glycan structures are elucidated in a single MS experiment. However, to date there is not a consensus regarding which mass spectrometric platform provides the best glycosylation coverage information. Herein, we employ two of the most widely used MS approaches, online high performance liquid chromatography-electrospray ionization mass spectrometry (HPLC/ESI-MS) and offline HPLC followed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), to determine which of the two approaches provides the best glycosylation coverage information of a complex glycoprotein, the group M consensus HIV-1 envelope, CON-S gp140DeltaCFI, which has 31 potential glycosylation sites. Our results highlight differences in the informational content obtained between the two methods such as the overall number of glycosylation sites detected, the numbers of N-linked glycans present at each site, and the type of confirmatory information obtained about the glycopeptide using MS/MS experiments. The two approaches are quite complementary, both in their coverage of glycopeptides and in the information they provide in MS/MS experiments. The information in this study contributes to the field of mass spectrometry by demonstrating the strengths and limitations of two widely used MS platforms in glycoprotein analysis.     

Mass spectrometry in the detection and diagnosis of congenital disorders of glycosylation

Mass spectrometry (MS) of large molecules such as proteins and oligosaccharides has not been employed in clinical practices, while that of small metabolites is widely used for the screening and diagnosis of various congenital diseases. Congenital disorders of glycosylation (CDG) is a newly recognized group of diseases derived from defects in the biosynthetic pathway of protein glycosylation and the patients are never decisively diagnosed unless the glycoprotein molecules are analyzed. We have constructed a diagnostic system where MS of glycoproteins and glycopeptides identifies abnormalities in their glycan moieties. This program is anticipated to reveal the prevalence of CDG and to demonstrate the essential role of MS in the emerging field of medicine, disease glycomics and glycoproteomics.     

Characterization of oligosaccharide moieties of intact glycoproteins by microwave-assisted partial acid hydrolysis and mass spectrometry

A method, which utilizes microwave-assisted partial acid hydrolysis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS), to elucidate oligosaccharide composition of intact glycoproteins is presented here. Glycoproteins, such as ribonuclease B, avidin, alpha1-acid glycoprotein, and fetuin, are used as model systems to demonstrate this technique. Partial cleavage of oligosaccharides from whole intact glycoproteins with trifluoroacetic acid was observed after a short exposure to microwaves. Due to the high-resolution mass spectra obtained by MALDI-TOFMS from glycoproteins with molecular weights less than 20 kDa, the compositions of oligosaccharides are readily derived for ribonuclease B and avidin. The data agree with the proposed oligosaccharide structures of ribonuclease B (five glycoforms) and avidin (eight glycoforms). Larger glycoproteins such as alpha1-acid glycoprotein (many glycoforms) and fetuin (many glycoforms) exhibited only broad peaks with no glycoform resolution. Nevertheless, this method can be used successfully for analysis of glycoproteins with molecular weights greater than 20 kDa to determine the presence or absence of glycosylation.     

Improvement of mass spectrometry analysis of glycoproteins by MALDI-MS using 3-aminoquinoline/α-cyano-4-hydroxycinnamic acid

Protein glycosylation analysis is important for elucidating protein function and molecular mechanisms in various biological processes. We previously developed a glycan analysis method using a 3-aminoquinoline/α-cyano-4-hydroxycinnamic acid liquid matrix (3-AQ/CHCA LM) and applied it to the quantitative glycan profiling of glycoproteins. However, information concerning glycosylation sites is lost; glycopeptide analysis is therefore required to identify the glycosylation sites in glycoproteins. Human epidermal growth factor receptor 2 (HER2) is a glycoprotein that plays a role in the regulation of cell proliferation, differentiation, and migration. Several reports have described the structure of HER2, but the structures of N-glycans attached to this protein remain to be fully elucidated. In this study, 3-AQ/CHCA LM was applied to tryptic digests of HER2 to reveal its N-glycosylation state and to evaluate the utility of this LM in characterizing glycopeptides. Peptide sequence coverage was considerably improved compared to analysis of HER2 using α-cyano-4-hydroxycinnamic acid or 2,5-dihydroxybenzoic acid. Most of the peaks observed using only this LM were localized at the inner or outer regions of sample spots. Furthermore, five of the peptide peaks that were enriched within the inner region were confirmed to be glycosylated by MS/MS analysis. Three glycosylation sites were identified and their glycan structures were elucidated. The reduction in sample complexity by on-target separation allowed for higher sequence coverage, resulting in effective detection and characterization of glycopeptides. In conclusion, these results demonstrate that MS-based glycoprotein analysis using 3-AQ/CHCA is an effective method to identify glycosylation sites in proteins and to elucidate the glycan structures of glycoproteins in complex samples.     

Selective in vitro glycosylation of recombinant proteins: semi-synthesis of novel homogeneous glycoforms of human erythropoietin

BACKGROUND: A natural glycoprotein usually exists as a spectrum of glycosylated forms, where each protein molecule may be associated with an array of oligosaccharide structures. The overall range of glycoforms can have a variety of different biophysical and biochemical properties, although details of structure-function relationships are poorly understood, because of the microheterogeneity of biological samples. Hence, there is clearly a need for synthetic methods that give access to natural and unnatural homogeneously glycosylated proteins. The synthesis of novel glycoproteins through the selective reaction of glycosyl iodoacetamides with the thiol groups of cysteine residues, placed by site-directed mutagenesis at desired glycosylation sites has been developed. This provides a general method for the synthesis of homogeneously glycosylated proteins that carry saccharide side chains at natural or unnatural glycosylation sites. Here, we have shown that the approach can be applied to the glycoprotein hormone erythropoietin, an important therapeutic glycoprotein with three sites of N-glycosylation that are essential for in vivo biological activity. RESULTS: Wild-type recombinant erythropoietin and three mutants in which glycosylation site asparagine residues had been changed to cysteines (His(10)-WThEPO, His(10)-Asn24Cys, His(10)-Asn38Cys, His(10)-Asn83CyshEPO) were overexpressed and purified in yields of 13 mg l(-1) from Escherichia coli. Chemical glycosylation with glycosyl-beta-N-iodoacetamides could be monitored by electrospray MS. Both in the wild-type and in the mutant proteins, the potential side reaction of the other four cysteine residues (all involved in disulfide bonds) were not observed. Yield of glycosylation was generally about 50% and purification of glycosylated protein from non-glycosylated protein was readily carried out using lectin affinity chromatography. Dynamic light scattering analysis of the purified glycoproteins suggested that the glycoforms produced were monomeric and folded identically to the wild-type protein. CONCLUSIONS: Erythropoietin expressed in E. coli bearing specific Asn-->Cys mutations at natural glycosylation sites can be glycosylated using beta-N-glycosyl iodoacetamides even in the presence of two disulfide bonds. The findings provide the basis for further elaboration of the glycan structures and development of this general methodology for the synthesis of semi-synthetic glycoproteins.     

香豆素类新基质在基质辅助激光解吸／电离飞行时间质谱测定多糖和糖蛋白中的应用

将几种香豆素类新基质(香豆素、3-羟基香豆素(3-HC)、3-氨基香豆素(3-AC)、3-羧基香豆素(3-CC)和4-甲基-7-羟基香豆素(4-M-7-HC))分别应用于基质辅助激光解吸/电离飞行时间质谱(MALDI TOF-MS)测定葡聚糖和3种糖蛋白的研究.香豆素和3-羟基香豆素分别与2,5-二羟基苯甲酸(DHB)混合组成2种二元基质,极大地改善了基质和葡聚糖样品的共结晶状况,样品分布更加均匀.葡聚糖样品更易解吸/电离,每个激光点照射样品均能产生较强的质谱信号,且谱图重现性更好,得到了理想的MALDI TOF-MS谱图.当香豆素类基质用于分析糖蛋白时:3-HC和4-M-7-HC是测定糖蛋白A的优异基质,能检测到m/z 为66 672 Da 的离子信号.而3-AC测定糖蛋白B的基质效果比糖类分析常用基质2,5-二羟基苯甲酸更好.因此,这些香豆素类化合物将为MALDI TOF-MS分析多糖和糖蛋白提供更多新基质选择.     

Isolation and immunological characterization of an iron-regulated, transformation-sensitive cell surface protein of normal rat kidney cells.

We have analyzed the surfacr proteins of cultured normal rat kidney (NRK) cells and virus-transformed NRK cells subjected to iron deprivation. Such a treatment specifically induces two transformation-sensitive plasma membrane-associated glycoproteins with a subunit molecular weight of 160,000 (160 K) and 130,000 (130 K) daltons in NRK cells. In these cells the 160 K glycoprotein is readily available to lactoperoxidase-mediated iodination, and the 130 K is apparently inaccessible to iodination. Major differences were revealed when iodinated membrane proteins of normal and virus-transformed cells subjected to iron deprivation were compared. In Kirsten sarcoma virus-transformed NRK cells the 160 K glycoprotein was weakly labeled. In two clones of simian virus 40-transformed NRK cells the 160 K glycoprotein was weakly labeled or not at all. The 130 K glycoprotein was inaccessible to iodination in all virus-transformed cell lines. The 160 K and 130 K glycoproteins were isolated from plasma membranes of NRK cells using preparative SDS gel electrophoresis. Antibodies generated against these glycoproteins stained the external surfaces of NRK cells and induced antigen redistribution. Evidence presented suggests that 160 K and 130 K are plasma membrane-associated procollagen molecules. A possible interaction of these proteins with transferrin is also described. The data suggest that these proteins may have an important role in the sequence of events leading to transformation.     

An approach to remove alpha amylase for proteomic analysis of low abundance biomarkers in human saliva

Proteomic characterization of human whole saliva for the identification of disease-specific biomarkers is guaranteed to be an easy-to-use and powerful diagnostic tool for defining the onset, progression and prognosis of human systemic diseases and, in particular, oral diseases. The high abundance of proteins, mainly alpha amylase, hampers the detection of low abundant proteins appearing in the disease state and therefore should be removed. In the present study a 2-DE was used to analyze human whole saliva following the removal of alpha amylase by affinity adsorption to potato starch. After alpha amylase removal whole saliva was analyzed by SDS-PAGE showing at least sixfold removal efficiency and by an alpha amylase activity assay showing 97% reduced activity. MS identification of the captured alpha amylase after elution demonstrated specific removal; 2-DE analysis showed the selective removal of alpha amylase and consequently increased gel resolution. MS identification of protein spots in the 60 kDa area revealed 15 proteins, which were masked before alpha amylase removal. In conclusion, treatment of human whole saliva with an alpha amylase removal device increases gel resolution and enables a higher protein sample for analysis.     

基于凝集素的唾液糖蛋白分离、分析及应用

唾液中的糖蛋白丰度偏低,给分离、分析带来挑战。该文采用麦胚素（WGA）和橙黄网胞盘菌凝集素（AAL）分别富集糖蛋白,考察了高丰度蛋白质去除和不同酶解方式对糖蛋白分离、分析的影响。结果显示,WGA和AAL提取的唾液糖蛋白经胶内酶解可鉴定到的糖蛋白数量显著多于溶液内酶解的结果,也优于去除高丰度蛋白质后的鉴定结果。选择WGA结合胶内酶解进一步对比分析肺癌患者与健康人唾液糖蛋白的差异,通过免标记定量分析共鉴定到139个蛋白质,其中102个蛋白质存在糖基化位点,包括14个在癌症组和正常组之间存在显著差异（p<0.05）的糖蛋白,表明该策略可用于唾液糖蛋白的有效分离、分析和癌症标志物的发现。