Discrimination of Aspergillus isolates at the species and strain level by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry fingerprinting

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF MS) was used to generate highly reproducible mass spectral fingerprints for 12 species of fungi of the genus Aspergillus and 5 different strains of Aspergillus flavus. Prior to MALDI–TOF MS analysis, the fungi were subjected to three 1-min bead beating cycles in an acetonitrile/trifluoroacetic acid solvent. The mass spectra contain abundant peaks in the range of 5 to 20 kDa and may be used to discriminate between species unambiguously. A discriminant analysis using all peaks from the MALDI–TOF MS data yielded error rates for classification of 0 and 18.75% for resubstitution and cross-validation methods, respectively. If a subset of 28 significant peaks is chosen, resubstitution and cross-validation error rates are 0%. Discriminant analysis of the MALDI–TOF MS data for 5 strains of A. flavus using all peaks yielded error rates for classification of 0 and 5% for resubstitution and cross-validation methods, respectively. These data indicate that MALDI–TOF MS data may be used for unambiguous identification of members of the genus Aspergillus at both the species and strain levels.     

Application of targeted quantitative proteomics analysis in human cerebrospinal fluid using a liquid chromatography matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometer (LC MALDI TOF/TOF) platform

Targeted quantitative proteomics by mass spectrometry aims to selectively detect one or a panel of peptides/proteins in a complex sample and is particularly appealing for novel biomarker verification/validation because it does not require specific antibodies. Here, we demonstrated the application of targeted quantitative proteomics in searching, identifying, and quantifying selected peptides in human cerebrospinal spinal fluid (CSF) using a matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometer (MALDI TOF/TOF)-based platform. The approach involved two major components: the use of isotopic-labeled synthetic peptides as references for targeted identification and quantification and a highly selective mass spectrometric analysis based on the unique characteristics of the MALDI instrument. The platform provides high confidence for targeted peptide detection in a complex system and can potentially be developed into a high-throughput system. Using the liquid chromatography (LC) MALDI TOF/TOF platform and the complementary identification strategy, we were able to selectively identify and quantify a panel of targeted peptides in the whole proteome of CSF without prior depletion of abundant proteins. The effectiveness and robustness of the approach associated with different sample complexity, sample preparation strategies, as well as mass spectrometric quantification were evaluated. Other issues related to chromatography separation and the feasibility for high-throughput analysis were also discussed. Finally, we applied targeted quantitative proteomics to analyze a subset of previously identified candidate markers in CSF samples of patients with Parkinson's disease (PD) at different stages and Alzheimer's disease (AD) along with normal controls.     

The lipid composition of the unicellular green alga Chlamydomonas reinhardtii and the diatom Cyclotella meneghiniana investigated by MALDI-TOF MS and TLC

The lipid composition of algae is crucial for numerous structural and physiological aspects, e.g. the integrity of the photosynthetic complexes and the functionality of membrane-embedded processes as the photosynthetic electron transport in thylakoids or the mitochondrial respiration. In this paper the lipid composition of the organic extracts of the green alga Chlamydomonas reinhardtii and the diatom Cyclotella meneghiniana are compared by using matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) in combination with thin-layer chromatography (TLC). The combined methods enable quantitative evaluation of the individual lipid classes as well as the determination of the relative acyl compositions. It will be shown that both algae differ in (a) the lipid classes, (b) the relative contribution of the individual lipid classes and (c) the acyl compositions. Differences in the acyl composition concern particularly the mono- and digalactosyl diacylglycerols. Glycerol-trimethylhomoserine and phosphatidylethanolamine are exclusively detected in the C. reinhardtii extracts, whereas phosphatidylcholine is a characteristic lipid of C. meneghiniana. Furthermore, the proportion of the acidic lipids sulfoquinovosyl-diacylglycerol and phosphatidylglycerol is significantly higher in the diatom than in C. reinhardtii.     

Detection of protein modifications and counterfeit protein pharmaceuticals using isotope tags for relative and absolute quantification and matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry: studies of insulins

Isotope tags for relative and absolute quantification (iTRAQ) reagent coupled with matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI-TOF/TOF) mass spectrometric analysis has been evaluated as both a qualitative and quantitative method for the detection of modifications to active pharmaceutical ingredients derived from recombinant DNA technologies and as a method to detect counterfeit drug products. Five types of insulin (human, bovine, porcine, Lispro, and Lantus) were used as model products in the study because of their minor variations in amino acid sequence. Several experiments were conducted in which each insulin variant was separately digested with Glu-C, and the digestate was labeled with one of four different iTRAQ reagents. All digestates were then combined for desalting and MALDI-TOF/TOF mass spectrometric analysis. When the digestion procedure was optimized, the insulin sequence coverage was 100%. Five different types of insulin were readily differentiated, including human insulin (P28K29) and Lispro insulin (K28P29), which differ only by the interchange of two contiguous residues. Moreover, quantitative analyses show that the results obtained from the iTRAQ method agree well with those determined by other conventional methods. Collectively, the iTRAQ method can be used as a qualitative and quantitative technique for the detection of protein modification and counterfeiting.     

Microwave-assisted sample preparation for rapid and sensitive analysis of H. pylori lipid A applicable to a single colony

The lipid A of Gram-negative bacteria plays a major role in the pathogenesis of bacterial infections. Lipid A diversity is observed both in the number and length of fatty-acid side chains and in the presence of terminal phosphate residues and associated modifications. In this report, we describe a new sample preparation method based on microwave-assisted enzymatic digestion and detergent-free mild hydrolysis, in conjunction with a MALDI-time-of-flight (TOF)/TOF analysis, to determine the structures of lipid A from Helicobacter pylori. The total time for sample preparation and mass spectrometric analysis is within 2 h and applicable to profiling the lipid A structures from dried bacterial cells on as little as 1 μg. The reliability of the technique was further demonstrated through the analysis of the lipid A from bacterial cells of different H. pylori strains. The phosphorylation and acylation patterns of lipid A could be elucidated using material from a single colony. Furthermore, we found unusual heptaacyl lipid A species present in H. pylori mutant that have not been previously reported, although the abundance was relatively low. The present study provides the first characterization of the lipid A component from a single bacterial colony sample by mass spectrometry.     

Protein N-glycosylation is similar in the moss Physcomitrella patens and in higher plants

We have investigated the structure of glycans N-linked to the proteins of the moss Physcomitrella patens. The structural elucidation was carried out by western blotting using antibodies specific for N-glycan epitopes and by analysis of N-linked glycans enzymatically released from a total protein extract by combination of MALDI–TOF and MALDI–PSD mass spectrometry analysis. Nineteen N-linked oligosaccharides were characterised ranging from high-mannose-type and truncated paucimannosidic-type to complex-type N-glycans harbouring core-xylose, core-(1,3)-fucose and Lewis^a, as previously described for proteins from higher plants. This demonstrates that the processing of N-linked glycans, as well as the specificity of glycosidases and glycosyltransferases involved in this processing, are highly conserved between P. patens and higher plants. As a consequence, P. patens appears to be a new promising model organism for the investigation of the biological significance of protein N-glycosylation in the plant kingdom, taking advantage of the potential for gene targeting in this moss.Abbreviations Asn asparagine - CID collision-induced dissociation - Glc glucose - GlcNAc N-acetylglucosamine - Man mannose - MALDI–TOF MS matrix-assisted laser desorption ionization–time of flight mass spectrometry - PNGase A peptide N-glycosidase A - PSD post-source decay     

Purification and properties of two blue biliproteins from the larval hemolymph and integument of Rhodinia fugax (Lepidoptera: Saturniidae)

Blue biliproteins (BPs) are found in the hemolymph and integument of the fifth instar larvae of the saturniid silkworm, Rhodinia fugax. An efficient method of isolating BPs from the hemolymph, epidermis and cuticle using hydrophobic interaction chromatography and ion-exchange chromatography was devised. The BPs from the hemolymph, epidermis and cuticle have molecular weights of approximately 24 000, 48 000 and 23 000 Da by gel-filtration, respectively. Using matrix-assisted laser desorption ionization–time of flight/mass spectrometry (MALDI–TOF/MS), the respective molecular masses were determined to be 22 641, 22 908 and 22 737 Da. Based on these results, BP molecules from the hemolymph and cuticle are assumed to be monomers, whereas the epidermal BP is a dimer. The amino acid composition and N-terminal amino acid sequences of the BPs from the hemolymph and cuticle (BP-I) are very similar, but the BP from the epidermis (BP-II) is quite different. The N-terminal amino acid sequences of these BPs share approximately 50% identity with the biliproteins from other lepidopteran insects. The blue color of BP is due to the presence of bile pigments, which are non-covalently bound to the apoprotein. The absorbance spectrum of BP-I from the hemolymph revealed maxima at 280 and 669 nm, while that of BP-II showed maxima at 280, 385 and 663 nm. The pigment dimethyl esters were extracted from BP-I and BP-II with acidic methanol and dichloromethane. The results of these analyses suggest that the blue pigments of BP-I and BP-II are different; BP-I contains a phorcabilin-like pigment while BP-II contains biliverdin IXγ. In an immunoblot analysis, anti-BP-I antibodies, produced against hemolymph BP-I, reacted with immunoreactive proteins in the hemolymph and cuticle of R. fugax. These anti-BP-I antibodies did not react with BP-II and only cross-reacted weakly with Samia cynthia ricini biliverdin-binding protein (BBP)-II.     

Protein labeling by iTRAQ: a new tool for quantitative mass spectrometry in proteome research

A novel, MS-based approach for the relative quantification of proteins, relying on the derivatization of primary amino groups in intact proteins using isobaric tag for relative and absolute quantitation (iTRAQ) is presented. Due to the isobaric mass design of the iTRAQ reagents, differentially labeled proteins do not differ in mass; accordingly, their corresponding proteolytic peptides appear as single peaks in MS scans. Because quantitative information is provided by isotope-encoded reporter ions that can only be observed in MS/MS spectra, we analyzed the fragmentation behavior of ESI and MALDI ions of peptides generated from iTRAQ-labeled proteins using a TOF/TOF and/or a QTOF instrument. We observed efficient liberation of reporter ions for singly protonated peptides at low-energy collision conditions. In contrast, increased collision energies were required to liberate the iTRAQ label from lysine side chains of doubly charged peptides and, thus, to observe reporter ions suitable for relative quantification of proteins with high accuracy. We then developed a quantitative strategy that comprises labeling of intact proteins by iTRAQ followed by gel electrophoresis and peptide MS/MS analyses. As proof of principle, mixtures of five different proteins in various concentration ratios were quantified, demonstrating the general applicability of the approach presented here to quantitative MS-based proteomics.     

Epitope mapping of the gastrin-releasing peptide/anti-bombesin monoclonal antibody complex by proteolysis followed by matrix-assisted laser desorption ionization mass spectrometry.

We have developed a method to rapidly identify the antigenic determinant for an antibody using in situ proteolysis of an immobilized antigen-antibody complex followed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI/TOF). A mouse anti-bombesin monoclonal antibody was immobilized to agarose beads and then the antigen, gastrin-releasing peptide (GRP), was allowed to bind. Direct analysis of the immobilized antigen-antibody complex by MALDI/TOF is demonstrated and allows identification of ca. 1 pmol of the bound GRP. To identify the epitope, the immobilized antigen-antibody complex was subjected to proteolysis with trypsin, chymotrypsin, thermolysin, and aminopeptidase M. Following proteolysis, the part of the antigen in contact with the antibody and protected from proteolysis was identified directly by MALDI/TOF. Subsequently, the epitope was eluted from the immobilized antibody with 0.1 M glycine buffer (pH 2.3), separated by reversed-phase HPLC, and its identity confirmed by MALDI/TOF. Using this approach, the epitope for the anti-bombesin monoclonal antibody was shown to comprise the last 7-8 residues (HWAVGHLM-NH2) of GRP.     

Physiological levels of diacylglycerols in phospholipid membranes induce membrane fusion and stabilize inverted phases

In the preceding paper (Ellens et al., 1989), it was shown that liposome fusion rates are substantially enhanced under the same conditions which induce isotropic 31P NMR resonances in multilamellar dispersions of the same lipid. Both of these phenomena occur within the same temperature interval, delta TI, below the L alpha/HII phase transition temperature, TH. TH and delta TI can be extremely sensitive to the lipid composition. The present work shows that 2 mol% of diacylglycerols like those produced by the phosphatidylinositol cycle in vivo can lower TH, delta TI, and the temperature for fast membrane fusion by 15-20 degrees C. N-Monomethylated dioleoylphosphatidylethanolamine is used as a model system. These results show that physiological levels of diacylglycerols can substantially increase the susceptibility of phospholipid membranes to fusion. This suggests that, in addition to their role in protein kinase C activation, diacylglycerols could play a more direct role in the fusion event during stimulus-exocytosis coupling in vivo.     

Label-free mass spectrometry-based relative quantification of proteins separated by one-dimensional gel electrophoresis

Here we present a matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI–TOF/TOF)-based label-free relative protein quantification strategy that involves sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) separation of proteins followed by in-gel trypsin digestion. The main problem encountered in gel-based protein quantification is the difficulty in achieving complete and consistent proteolytic digestion. To solve this problem, we developed a high-pressure-assisted in-gel trypsin digestion method that is based on pressure cycling technology (PCT). The PCT approach performed at least as well as the conventional overnight in-gel trypsin digestion approach in parameters such as number of peaks detected, number of peptides identified, and sequence coverage, and the digestion time was reduced to 45 min. The gel/mass spectrometry (MS)-based label-free protein quantification method presented in this work proved the applicability of the signal response factor concept for relative protein quantification previously demonstrated by other groups using the liquid chromatography (LC)/MS platform. By normalizing the average signal intensities of the three most intense peptides of each protein with the average intensities of spiked synthetic catalase tryptic peptides, which we used as an internal standard, we observed spot-to-spot and lane-to-lane coefficients of variation of less than 10 and 20%, respectively. We also demonstrated that the method can be used for determining the relative quantities of proteins comigrating during electrophoretic separation.     

Strategy combining separation of isotope-labeled unfolded proteins and matrix-assisted laser desorption/ionization mass spectrometry analysis enables quantification of a wide range of serum proteins

A novel strategy for the quantitative profiling of serum proteome is described. It includes an ammonium sulfate depletion of the serum, an affordable stable isotope labeling chemistry for samples with a large amount of protein, separation of the unfolded proteins, and relative quantification by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS). Labeling of unfolded proteins was performed using normal (D₀) acrylamide and deuterated (D₃) acrylamide. The workflow for separating the unfolded proteins includes whole gel elution and ion exchange liquid chromatography, and it combines electrophoretic separation based on the protein molecular weight followed by chromatographic separation in the presence of 8 M urea based on protein charge. This was followed by trypsinolysis and MALDI MS analysis, leading to the quantification of a large number of serum proteins, including those with an abundance of 10^-5 less than albumin. This robust and inexpensive workflow is suitable for the quantitative profiling of protein changes in serum associated with preanalytical variables.     

High-throughput phospholipid quantitation in mammalian cells using matrix-assisted laser desorption ionization-time of flight mass spectrometry with N-trifluoroacetyl-phosphatidylethanolamine as internal standard

A high-throughput method is described for quantitative analysis of phospholipids. The method comprises extraction of lipids, addition of the internal standard N-trifluoroacetyl-phosphatidylethanolamine, and final analysis using matrix-assisted laser desorption ionization mass spectrometry. Quantitative data are obtained by calibration directly in the sample matrix. Calibration with one phosphatidylcholine was found sufficient for quantification of all major phosphatidylcholines tested. The method is very sensitive, has broad application, and is easily applicable to any biological sample. The detection limit for phosphatidylcholines was clearly below 2 μg on the spot, requiring less than 4000 cells corresponding to about 1.6 μg cell dry mass.     

Biochemical, molecular, and functional characterization of PISCF-allatostatin, a regulator of juvenile hormone biosynthesis in the mosquito Aedes aegypti

Aedes aegypti PISCF-allatostatin or allatostatin-C (Ae-AS-C) was isolated using a combination of high performance liquid chromatography and enzyme-linked immunosorbent assay (ELISA). The matrix-assisted laser desorption/ionization time-of-flight (TOF) mass spectrum of positive ELISA fractions revealed a molecular mass of 1919.0 Da, in agreement with the sequence qIRYRQCYFNPISCF, with bridged cysteines. This sequence was confirmed by matrix-assisted laser desorption/ionization tandem TOF/TOF mass spectrometry analysis. The corresponding Ae-AS-C cDNA was amplified by PCR, and the sequence of the peptide was confirmed. An in vitro radiochemical assay was used to study the inhibitory effect of synthetic Ae-AS-C on juvenile hormone biosynthesis by the isolated corpora allata (CA) of adult female A. aegypti. The inhibitory action of synthetic Ae-AS-C was dose-dependent; with a maximum at 10(-9) m. Ae-AS-C showed no inhibitory activity in the presence of farnesoic acid, an immediate precursor of juvenile hormone, indicating that the Ae-AS-C target is located before the formation of farnesoic acid in the pathway. The sensitivity of the CA to inhibition by Ae-AS-C in the in vitro assay varied during the adult life; the CA was most sensitive during periods of low synthetic activity. In addition, the levels of Ae-AS-C in the brain were studied using ELISA and reached a maximum at 3 days after eclosion. These studies suggest that Ae-AS-C is an important regulator of CA activity in A. aegypti.     

Glass-supported lipid/polydiacetylene films for colour sensing of membrane-active compounds

Glass-supported biomimetic lipid/polydiacetylene films were employed for colourimetric detection and analysis of amphiphilic and membrane-active molecules. The sensor films comprise lipid monolayers that constitute a biomimetic membrane platform, interspersed within polydiacetylene domains that function as the colour reporter. The optical detection scheme is based on visible blue–red transitions of polydiacetylene, induced by amphiphilic analytes interacting with the film. The colour transitions of the lipid/polydiacetylene films can be either detected by the naked eye, recorded spectroscopically, or registered through digital image analysis using conventional scanning devices. Digital image analysis, in particular, allows quantification of the colourimetric transformations. Detection threshold of micromolar concentration of a membrane-active cytolytic peptide is demonstrated.     

Lipidomic characterization of exosomes isolated from human plasma using various mass spectrometry techniques

Ultrahigh-performance supercritical fluid chromatography - mass spectrometry (UHPSFC/MS), ultrahigh-performance liquid chromatography - mass spectrometry (UHPLC/MS), and matrix-assisted laser desorption/ionization (MALDI) - MS techniques were used for the lipidomic characterization of exosomes isolated from human plasma. The high-throughput methods UHPSFC/MS and UHPLC/MS using a silica-based column containing sub-2 μm particles enabled the lipid class separation and the quantitation based on exogenous class internal standards in <7 minute run time. MALDI provided the complementary information on anionic lipid classes, such as sulfatides. The nontargeted analysis of 12 healthy volunteers was performed, and absolute molar concentration of 244 lipids in exosomes and 191 lipids in plasma belonging to 10 lipid classes were quantified. The statistical evaluation of data included principal component analysis, orthogonal partial least square discriminant analysis, S-plots, p-values, T-values, fold changes, false discovery rate, box plots, and correlation plots, which resulted in the information on lipid changes in exosomes in comparison to plasma. The major changes were detected in the composition of triacylglycerols, diacylglycerols, phosphatidylcholines, and lysophosphatidylcholines, whereby sphingomyelins, phosphatidylinositols, and sulfatides showed rather similar profiles in both biological matrices.     

小鼠晶体蛋白质组的双向电泳和质谱鉴定研究

目的应用双向电泳和质谱技术研究5周龄小鼠晶体蛋白质组。方法提取小鼠晶体总蛋白,进行固相pH梯度（IPG）等电聚焦双向电泳,胶体考马斯亮蓝R-250染色,使用PDQuest7．30图像分析软件分析电泳图像。选择主要蛋白点胶上酶解,应用基质辅助激光解析电离飞行时间／飞行时间（MALDI—TOF／TOF）仪器进行串联质谱（MS／MS）鉴定。结果上样量为882μg和190μg时,分别检测370±41蛋白点（n=3）和57±5个蛋白点（n=3）。高上样量能够较好地分离晶体低丰度蛋白,如念珠状纤维结构蛋白BFSP;低上样量可很好地分离高丰度蛋白-晶体蛋白（包括αA、αB;βA1～βA4;βB1～βB3;γA～γF和γS等）。质谱鉴定得到1种细胞骨架蛋白和16种高丰度晶体蛋白。结论双向电泳和质谱技术有效考察了晶体总蛋白质,为分析白内障形成过程中蛋白质的表达改变提供了新的方法和途径。     

Comprehensive analysis of the major lipid classes in sebum by rapid resolution high-performance liquid chromatography and electrospray mass spectrometry

Sebum is a complex lipid mixture that is synthesized in sebaceous glands and excreted on the skin surface. The purpose of this study was the comprehensive detection of the intact lipids that compose sebum. These lipids exist as a broad range of chemical structures and concentrations. Sebum was collected with SebuTape^TM from the foreheads of healthy donors, and then separated by HPLC on a C8 stationary phase with sub 2 µm particle size. This HPLC method provided high resolution and excellent reproducibility of retention times (RT). Compound mining was performed with time of flight (TOF) and triple quadrupole (QqQ) mass spectrometers (MS), which allowed for the classification of lipids according to their elemental composition, degree of unsaturation, and MS/MS fragmentation. The combination of the two MS systems detected 95 and 29 families of triacylglycerols (TAG) and diacylglycerols (DAG), respectively. Assignment was carried out regardless of positional isomerism. Among the wax esters (WE), 28 species were found to contain the 16:1 fatty acyl moiety. This method was suitable for the simultaneous detection of squalene and its oxygenated derivative. A total of 9 cholesterol esters (CE) were identified and more than 48 free fatty acids (FFA) were detected in normal sebum. The relative abundance of each individual lipid within its own chemical class was determined for 12 healthy donors. In summary, this method provided the first characterization of the features and distribution of intact components of the sebum lipidome.     

Age-dependent loss of seed viability is associated with increased lipid oxidation and hydrolysis

The accumulation of reactive oxygen species has been associated with a loss of seed viability. Therefore, we have investigated the germination ability of a range of seed stocks, including two wheat collections and one barley collection that had been dry-aged for 5–40 years. Metabolite profiling analysis revealed that the accumulation of glycerol was negatively correlated with the ability to germinate in all seed sets. Furthermore, lipid degradation products such as glycerol phosphates and galactose were accumulated in some seed sets. A quantitative analysis of nonoxidized and oxidized lipids was performed in the wheat seed set that showed the greatest variation in germination. This analysis revealed that the levels of fully acylated and nonoxidized storage lipids like triacylglycerols and structural lipids like phospho- and galactolipids were decreasing. Moreover, the abundance of oxidized variants and hydrolysed products such as mono-/diacylglycerols, lysophospholipids, and fatty acids accumulated as viability decreased. The proportional formation of oxidized and nonoxidized fatty acids provides evidence for an enzymatic hydrolysis of specifically oxidized lipids in dry seeds. The results link reactive oxygen species with lipid oxidation, structural damage, and death in long-term aged seeds.     

Mass spectrometric quantification of neutral and sialylated N-glycans from a recombinant therapeutic glycoprotein produced in the two Chinese hamster ovary cell lines

Quality control and assurance of glycan profiles of a recombinant glycoprotein from lot to lot is a critical issue in the pharmaceutical industry. To develop an easy and simple quantitative and qualitative glycan profile method based on matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS), the modification with Girard’s reagent T (GT) was exploited. Because GT-derivatized quantification of oligosaccharides using MALDI-TOF MS is possible only with neutral glycans, sialylated glycans are not subjected to quantitative analysis with MALDI-TOF MS. To solve this problem, mild methyl esterification and subsequent GT derivatization were employed, enabling us to perform rapid qualitative and quantitative analysis of sialylated and neutral N-linked oligosaccharides using MALDI-TOF MS. This modified method was used in the comparative quantification of N-glycans from the recombinant therapeutic glycoprotein expressed in two different Chinese hamster ovary (CHO) cell lines. The percentages of sialylated N-glycans to total were 22.5 and 5.2% in CHO-I and CHO-II cells, respectively, resulting in a significant difference in the biological activity of the recombinant glycoprotein.