N-glycan profiling of tissue samples to aid breast cancer subtyping.

Breast cancer is a highly heterogeneous disease. Its intrinsic subtype classification for diagnosis and choice of therapy traditionally relies on the presence of characteristic receptors. Unfortunately, this classification is often not sufficient for precise prediction of disease prognosis and treatment efficacy. The N-glycan profiles of 145 tumors and 10 healthy breast tissues were determined using Matrix-Assisted Laser Desorption-Ionization Time-of-Flight Mass Spectrometry. The tumor samples were classified into Mucinous, Lobular, No-Special-Type, Human Epidermal Growth Factor 2 + , and Triple-Negative Breast Cancer subtypes. Statistical analysis was conducted using the reproducibility-optimized test statistic software package in R, and the Wilcoxon rank sum test with continuity correction. In total, 92 N-glycans were detected and quantified, with 59 consistently observed in over half of the samples. Significant variations in N-glycan signals were found among subtypes. Mucinous tumor samples exhibited the most distinct changes, with 28 significantly altered N-glycan signals. Increased levels of tri- and tetra-antennary N-glycans were notably present in this subtype. Triple-Negative Breast Cancer showed more N-glycans with additional mannose units, a factor associated with cancer progression. Individual N-glycans differentiated Human Epidermal Growth Factor 2 + , No-Special-Type, and Lobular cancers, whereas lower fucosylation and branching levels were found in N-glycans significantly increased in Luminal subtypes (Lobular and No-Special-Type tumors). Clinically normal breast tissues featured a higher abundance of signals corresponding to N-glycans with bisecting moiety. This research confirms that histologically distinct breast cancer subtypes have a quantitatively unique set of N-glycans linked to clinical parameters like tumor size, proliferative rate, lymphovascular invasion, and metastases to lymph nodes. The presented results provide novel information that N-glycan profiling could accurately classify human breast cancer samples, offer stratification of patients, and ongoing disease monitoring.

Differentiation of Sialyl Linkages Using a Combination of Alkyl Esterification and Phenylhydrazine Derivatization: Application for N-Glycan Profiling in the Sera of Patients with Lung Cancer.

Alterations in oligosaccharides and types of sialic acid (SA) attachments have been associated with different pathological states. Matrix-assisted laser desorption mass spectrometry (MS) is commonly used for glycosylation studies. However, native sialylated glycans are suppressed or not detected during MS experiments. Consequently, different approaches have been employed to neutralize the negative charge of the carboxyl group. In this study, we present the advantage of phenylhydrazine (PHN) labeling for the detection and efficient discrimination of SA linkages when this derivatization follows alkyl esterification. As expected, PHN-labeled sialylated oligosaccharides with the 2,6-linkage type can be easily recognized according to the additional shift in mass corresponding to the presence of a methyl or ethyl group. Surprisingly, oligosaccharides with the 2,3-linked SA residue instead of a lactone were detected carrying the second PHN unit. This was beneficial as no further processing after esterification was needed to stabilize the lactone form. Moreover, during tandem mass experiments, all modified glycans produced favorable fragmentation patterns with a coherent recognition of SA linkages. Although both types of esterification, herein called the EST-PHN approach, provided comparable results, methylation exhibited marginally higher linkage specificity than ethyl esterification. The simplicity and effectiveness of the methodology are demonstrated on the model compound, sialyllactose, and its applicability for biological studies is presented on N-glycan profiling in the sera of lung cancer patients.

In vivo and in vitro cell-based model of lung adenocarcinoma from patients with pleural effusion.

Lung adenocarcinoma (LAC) is a common and aggressive form of lung cancer that is increasing in incidence among never smokers at a younger age. Current treatment of patients with LAC is insufficient and there is a need for identification of effective biomarkers and development of therapeutic targets. These demands require also improved models for in vivo and in vitro experimentation. In this study, we describe the establishment of two LAC cell lines, named LuCa-3 and LuCa-6. Both were derived from pleural effusion (PE) cells of LAC patients (L3 and L6) and readily propagated as tumor xenografts in immunodeficient mice. PE cells from the patient L6 exhibited also the capacity for in vitro growth and were cultured in two forms: (i) as a suspension growing cell population, labeled LuCa-6S, composed of non-clumping single cells; and (ii) as a monolayer-like culture, labeled LuCa-6A, exhibiting tight cell-to-cell and to culture surface adherence. Unique features of these two sublines and their cell clones are the capacity to convert from a non-clumping single-cell suspension into the adherent growth pattern and vice versa. Immunostaining of patients' tumor tissue xenografts and cultured subline cells displayed markers specific for the phenotype of human LAC. LuCa-6S and LuCa-6A cells did not reveal a noticeable disparity in quantitative growth characteristics. However, a number of differences were detected between these two cell populations manifested in detection or intensities of antigen expressions on the cell surface (CD133, SFTPC) and in the nucleus (TTF-1) including pluripotent (OCT-4, SOX-2, NANOG) genes in cancer stem-like cells (CSCs). Dissimilarities between these two sublines were also detected in N-glycan profiles and in the sensitivity to natural killer cells. Salient features of these subline cell populations are responsiveness to selective upregulation of the pluripotent genes in subsets of CSCs via conversion of their growth patterns and/or by using culture stem media with growth factors. The described in vivo/in vitro model enables broader experimental approaches in studies of lung adenocarcinoma.

N-Glycome changes reflecting resistance to platinum-based chemotherapy in ovarian cancer.

A number of studies have reported aberrant glycosylation in connection with malignancy. Our investigation further expands on this topic through the examination of N-glycans, which could be associated with the resistance of advanced stage, high-grade non-mucinous ovarian cancer to platinum/taxane based chemotherapy. We used tissue samples of 83 ovarian cancer patients, randomly divided into two independent cohorts (basic and validation). Both groups involved either cases with/without postoperative tumor residue or the cases determined either resistant or sensitive to this chemotherapy. In the validation cohort, preoperative serum samples were also available. N-glycans released from tumors and sera were permethylated and analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The MS analysis yielded a consecutive detection of 68 (tissue) and 63 (serum) N-glycan spectral signals. Eight of these were found to be differentially abundant in tissues of both independent cohorts including the cases with a postoperative cancer residue. One of these glycans was detected as differentially abundant in sera of the validation cohort. No statistically significant differences in intensities due to the same N-glycans were found in the cases without postoperative macroscopic residues in either the basic or validation cohort. From the biochemical point of view, the statistically significant N-glycans correspond to the structures carrying bisecting (terminal) GlcNAc residue and tetra-antennary structures with sialic acid and/or fucose residues. Among them, six tissue N-glycans could be considered potential markers connected with a resistance to chemotherapy in ovarian cancer patients. The prediction of primary resistance to standard chemotherapy may identify the group of patients suitable for alternative treatment strategies. SIGNIFICANCE: Drug resistance has become a major impediment to a successful treatment of patients with advanced ovarian cancer. The glycomic measurements related to cancer are becoming increasingly popular in identification of the key molecules as potential diagnostic and prognostic indicators. Our report deals with identification of differences in N-glycosylation of proteins in tissue and serum samples from the individuals showing sensitivity or resistance to platinum/taxane-based chemotherapy. The detection sensitivity to chemotherapy is vitally important for these patients.

Comprehensive N-glycosylation mapping of envelope glycoprotein from tick-borne encephalitis virus grown in human and tick cells.

Tick-borne encephalitis virus (TBEV) is the causative agent of severe human neuroinfections that most commonly occur after a tick bite. N-Glycosylation of the TBEV envelope (E) glycoprotein is critical for virus egress in mammalian cells, but not in tick cells. In addition, glycans have been reported to mask specific antigenic sites from recognition by neutralizing antibodies. In this regard, the main purpose of our study was to investigate the profile of N-glycans linked to the E protein of TBEV when grown in human neuronal cells and compare it to the profile of virus grown in tick cells. Mass spectrometric analysis revealed significant differences in these profiles. High-mannose glycan with five mannose residues (Man5GlcNAc2), a complex biantennary galactosylated structure with core fucose (Gal2GlcNAc2Man3GlcNAc2Fuc), and a group of hybrid glycans with the composition Gal0-1GlcNAc1Man3-5GlcNAc2Fuc0-1 were confirmed as the main asparagine-linked oligosaccharides on the surface of TBEV derived from human neuronal cells. The observed pattern was supported by examination of the glycopeptides, providing additional information about the glycosylation site in the E protein. In contrast, the profile of TBEV grown in tick cells showed that paucimannose (Man3-4 GlcNAc2Fuc0-1) and high-mannose structures with five and six mannoses (Man5-6GlcNAc2) were major glycans on the viral surface. The reported results complement existing crystallography and cryoelectron tomography data on the E protein structure and could be instrumental for designing carbohydrate-binding antiviral agents active against TBEV.

N-Glycan profiling of lung adenocarcinoma in patients at different stages of disease.

Lung adenocarcinoma (LAC) is the most common form of lung cancer that increases in non-smokers at younger age. Altered protein glycosylation is one of the hallmarks of malignancy, its role in cancer progression is still poorly understood. In this study, we report mass spectrometric (MS) analysis of N-glycans released from fresh or defrosted tissue specimens from 24 patients with LAC. Comparison of cancerous versus adjacent healthy tissues revealed substantial differences in N-glycan profiles associated with disease. The significant increase in paucimannose and high-mannose glycans with 6-9 mannose residues and decline in the sialylated complex biantenary core fucosylated glycan with composition NeuAcGal2GlcNAc2Man3GlcNAc2Fuc were general features of tumors. In addition, 42 new N-glycan compositions were detected in cancerous tissues. The prominent changes in advanced disease stages were mostly observed in core fucosylated N-glycans with additional fucose (Fuc) residue/s and enhanced branching with non-galactosylated N-acetyl-glucosamine (GlcNAc) units. Both of these monosaccharide types were linked preferably on the 6-antenna. Importantly, as compared with noncancerous tissues, a number of these significant changes were clearly detectable early on in stage I. Application of N-glycan data obtained from tissues was next assessed and validated for evaluation of small sized biopsies obtained via bronchoscopy. In summary, observed alterations and data of newly detected N-glycans expand knowledge about the glycosylation in LAC and may contribute to research in more tailored therapies. Moreover, the results demonstrate effectiveness of the presented approach for utility in rapid discrimination of cancerous from healthy lung tissues.

Applicability of Phenylhydrazine Labeling for Structural Studies of Fucosylated N-Glycans.

Fucosylation is a common modification, and its site in glycans refers to different normal and pathological processes. Despite intensive research, there is still a lack of methods to discriminate unambiguously the fucose position in one-step. In this work, we propose utility of phenylhydrazine (PHN) labeling for structural studies of fucosylated N-glycans by tandem MALDI mass spectrometry (MS) in the positive ion mode. PHN-tag influences the production of specific ion types, and the MS/MS fragmentation pattern provides useful structural information. All types of core fucosylated N-glycans have produced two abundant ions consistent with B- and C-glycosidic cleavages corresponding to the loss of the FucGlcNAcPHN residue with a mass 457 and 441 Da from the parent ions. These types of fragment ions in N-glycans without a core fucose were associated with the loss of the GlcNAcPHN unit (311 and 295 Da), and fucose cleavage followed the loss of the chitobiose residue. Since diagnostic useful cleavages produce peaks with significant intensities, this approach is also beneficial for rapid recognition of antenna from core fucosylation in glycans detected with low abundances. Moreover, in multifucosylated glycans, this type of labeling allows to distinguish how many fucose residues are on the specific antenna and provides additional information on the topology of N-glycans, such as type of antennarity or identification of bisecting moiety. The practical applicability of the approach is demonstrated on the analysis of multifucosylated N-glycans detected with lower abundances in lung cancer samples.

Efficient Procedure for N-Glycan Analyses and Detection of Endo H-Like Activity in Human Tumor Specimens.

Although the importance of glycosylation has been thoroughly recognized in association with a number of biological processes, efficient assessments of glycans have been hampered by both the limited size of specimens and lengthy sample preparations, particularly in clinical settings. Here we report a simple preparative method for N-glycan analyses. It involves only short one-step chloroform-methanol extraction in presence or absence of water prior to PNGase F deglycosylation. The procedure was successfully applied to the investigation of N-glycans obtained from small numbers of in vitro cultured cancer cells (≤1 × 10(5)) and to tumor tissues, including patient biopsies of small size. MALDI-MS analysis confirmed the efficient release of all N-glycan types including complex forms with poly-N-acetyllactosamine chains. In addition, nonaqueous extraction of specimens from several established cancer cell lines, as well as patient tumor tissues, yielded high-mannose glycans with one GlcNAc moiety (Man3-9GlcNAc), strongly suggesting preservation of enzymatic activity analogous to Endo H enzyme. In summary, the method is both a step toward the practical use of glycan profiling and a way to detect Endo H-like activity in cancer specimens.

The usefulness of hydrazine derivatives for mass spectrometric analysis of carbohydrates.

Over the last years, extensive studies have evaluated glycans from different biological samples and validated the importance of glycosylation as one of the most important post-translational modifications of proteins. Although a number of new methods for carbohydrate analysis have been published and there has been significant progress in their identification, the development of new approaches to study these biomolecules and understand their role in living systems are still vivid challenges that intrigue glycobiologists. In the last decade, the success in analyses of oligosaccharides has been driven mainly by the development of innovative, highly sensitive mass spectrometry techniques. For enhanced mass spectrometry detection, carbohydrate molecules are often derivatized. Besides, the type of labeling can influence the fragmentation pattern and make the structural analysis less complicated. In this regard, in 2003 we introduced the low scale, simple non-reductive tagging of glycans employing phenylhydrazine (PHN) as the derivatizing reagent. PHN-labeled glycans showed increased detection and as reported previously they can be analyzed by HPLC, ESI, or MALDI immediately after derivatization. Under tandem mass spectrometry conditions, PHN-derivatives produced useful data for the structural elucidation of oligosaccharides. This approach of analysis has helped to reveal new isomeric structures for glycans of known/unknown composition and has been successfully applied for the profiling of N-glycans obtained from serum samples and cancer cells. The efficacy of this labeling has also been evaluated for different substituted hydrazine reagents. This review summarizes all types of reducing-end labeling based on hydrazone-linkage that have been used for mass spectrometric analyses of oligosaccharides. This review is also aimed at correcting some past misconceptions or interpretations reported in the literature.

Alterations in glycopeptides associated with herceptin treatment of human breast carcinoma mcf-7 and T-lymphoblastoid cells.

The therapeutic humanized monoclonal antibody IgG1 known as Herceptin® has shown remarkable antitumor effects. Although this type of therapy has increased the cancer-free survival of patients, not all tumors respond to this treatment and cancers often develop resistance to the antibody. Despite the fact that Herceptin function has been extensively studied, the precise mechanism underlying its antitumor activity still remains incompletely defined. We previously demonstrated on human breast MCF-7 carcinoma and T-lymphoblastoid CEM cells that monoclonal antibody in combination with Lipoplex consisting of Lipofectamine mixed with plasmid DNA showed a more profound effect on cancer cell viability than antibody alone. The analyses of N-glycans isolated from cancer cells showed dramatic differences in profiles when cells were exposed to Herceptin. Moreover, the investigation of glycosylated peptides from the same cancer cell models after treatment revealed further alterations in the post-translational modifications. Tandem mass spectra obtained from the samples treated confirmed the presence of a series of glycopeptides bearing characteristic oligosaccharides as described in IgG1. However some of them differed by mass differences that corresponded to peptide backbones not described previously and more of them were detected from Herceptin treated samples than from cells transfected with Heceptin/Lipoplex. The results indicate that the presence of Lipoplex prevents antibody transformation and elongates its proper function. The better understanding of the multipart changes described in the glycoconjugates could provide new insights into the mechanism by which antibody induces regression in cancers.

α-D-mannose derivatives as models designed for selective inhibition of Golgi α-mannosidase II.

Human Golgi α-mannosidase II (hGM) is a pharmaceutical target for the design of inhibitors with anti-tumor activity. Nanomolar inhibitors of hGM exhibit unwanted co-inhibition of the human lysosomal α-mannosidase (hLM). Hence, improving specificity of the inhibitors directed toward hGM is desired in order to use them in cancer chemotherapy. We report on the rapid synthesis of D-mannose derivatives having one of the RS-, R(SO)- or R(SO(2))- groups at the α-anomeric position. Inhibitory properties of thirteen synthesized α-D-mannopyranosides were tested against the recombinant enzyme Drosophila melanogaster homolog of hGM (dGMIIb) and hLM (dLM408). Derivatives with the sulfonyl [R(SO(2))-] group exhibited inhibitory activities at the mM level toward both dGMIIb (IC(50) = 1.5-2.5 mM) and dLM408 (IC(50) = 1.0-2.0 mM). Among synthesized, only the benzylsulfonyl derivative showed selectivity toward dGMIIb. Its inhibitory activity was explained based on structural analysis of the built 3-D complexes of the enzyme with the docked compounds.

Contiguous O-galactosylation of 4(R)-hydroxy-l-proline residues forms very stable polyproline II helices.

The hydroxyproline-rich glycoproteins (HRGPs) are the major structural proteins of the extracellular matrix of algae and land plants. They are characterized by a rigid polyproline type II (PPII) conformation and extensive O-glycosylation of 4(R)-hydroxy-l-proline (Hyp) residues, which is a unique post-translational modification of proteins. The functional consequences of HRGP glycosylation remains unclear, but they have been implicated in contributing to their structural rigidity. Here, we have investigated the effects of naturally occurring beta-O-galactosylation of Hyp residues on the conformational stability of the PPII helix. In a series of well-defined model peptides Ac-(l-proline)(9)-NH(2) (1), Ac-(Hyp)(9)-NH(2) (2), and Ac-[Hyp(beta-d-galactose)](9)-NH(2) (3) we demonstrate that contiguous O-glycosylation of Hyp residues causes a dramatic increase in the thermal stability of the PPII helix according to analysis of thermal melting curves. This represents the first quantitative data on the contributions of glycosylation to stabilizing the PPII conformation. Molecular modeling indicates the increase in conformational stability may be due to a regular network of interglycan and glycan-peptide hydrogen bonds, in which the carbohydrate residues form a hydrophilic "overcoat" of the PPII helix. Evidence of this shielding effect of the amide backbone may be provided by analysis of the circular dichroism bands, which indicates an increase in the rho value of 3 relative to 1 and 2. This study gives further insight into the effects of naturally occurring Hyp beta-O-linked glycans on the PPII conformation as found in HRGPs in plant cell walls and also indicates that polyproline sequences may be suitable for the development of molecular scaffolds for the presentation of glycan structures.

Ex vivo assays of CEM cells cultured and treated in the three dimensional cultures.

This study was aimed at the applications of an ex vivo assays to characterization of CEM (Human T-Lymphoblastoid) cells. CEM cells were cultured in three dimensional (3-D) geometry in the Hollow Fibre Bioreactor (HFB) device. The cells were treated with Herceptin, anti-HER-2 (clone CB-11) and lipoplex containing lipofectamine (LipA) and plasmid DNA. To identify the response to treatment, the viability was established using Trypan blue assays. Magnetic resonance imaging (MRI) at 9.4Tesla (T) was applied for localization of the cells in the HFB device. The structural changes in the cells associated with treatment were examined with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The tryptic peptides and glycopeptides detected in treated cells provided evidence of the efficacy of antibody binding to the receptor. The results of the study confirmed that cells growth significantly decreased after treatment with antibodies and transfection with lipoplex.

N-glycomic changes in human breast carcinoma MCF-7 and T-lymphoblastoid cells after treatment with herceptin and herceptin/Lipoplex.

The humanized monoclonal antibody IgG1 in combination with chemotherapy has been demonstrated to enhance survival benefit in cancer treatment. Despite positive outcomes, some cancer cells develop multidrug resistance. Numerous mechanisms in cancers can be involved in the process of treatment therapy and most of them are not still well understood. To address how the carbohydrate moieties of cells are affected during treatment, the glycan profiles from the two most common cancer cell lines - human breast MCF-7 carcinoma and T-lymphoblastoid CEM cells - were studied here and compared with profiles after treatment with Herceptin alone or in combination with Lipofectamine mixed with plasmid DNA to form Lipoplex. N-Glycans were released from total cells by digestion with PNGaseF and analyzed by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). In summary, both original cell lines showed a dominant occurrence of high-mannose glycans. After treatment, these structures were suppressed and biantennary core-fucosylated glycans originating from IgG1 were the major carbohydrate products identified in cells. The high incidence of additional fucosylated or nonfucosylated galactosylated oligosaccharides, which were not detected in original cells or Herceptin, varied with conditions and time of exposure of cells to the antibody. The results presented in this study provide strong evidence for a role of glycosylation during antibody treatment.

Combined treatment of human MCF-7 breast carcinoma with antibody, cationic lipid and hyaluronic acid using ex vivo assays.

The effective targeting of malignant cell surface antigens is essential in cancer therapy. Resistance to treatment and rapid invasion of cancer cells are the main causes of cancer mortality. Despite intense research efforts, treatments often have demonstrated insufficient outcomes in clinical applications. The aim of the present study was to determine whether combined administration of monoclonal antibody (Herceptin, trastuzumab) and anti-HER-2 (clone CB11) with hyaluronic acid (HA) and lipoplex (containing lipofectamine (LipA) and plasmid DNA) can produce a synergistic reaction to increase the therapeutic effect of monoclonal antibodies. To assess the treatment response, we cultured a 3-D MCF-7 cell line overexpressing HER-2 and CD44 receptors. The high density 3-D cell aggregation in the hollow fiber bioreactor (HFB) used for the cell culture was monitored with the use of proton magnetic resonance imaging ((1)H MRI). In addition, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was used in combination with HPLC (high performance liquid chromatography) to evaluate structural changes in the proteins contained in treated cells. The study showed that incorporation of antibodies into targeted lipoplex results in more efficient delivery of the complex to tumor cells. The viability of cells decreased mostly due to cellular uptake of lipoplex and binding of the antibodies to the cellular surface receptor. The data also demonstrate that HA could be used to enhance treatment efficacy of trastuzumab and anti-HER-2 (clone CB11) in breast cancer cell cultures.

Mass spectrometric study of N-glycans from serum of woodchucks with liver cancer.

Woodchucks have been a preferred lab animal model of chronic hepatitis B viral infection. The model recapitulates the disease progression of HBV infection to hepatocellular carcinoma (HCC) and has documented similarities in protein glycosylation with human HCC. This study examined N-glycans in serum of animals with(out) HCC. Oligosaccharides were released enzymatically using PNGaseF from total serum or from serum partially fractionated by extraction. Two different extraction procedures - reversed-phase high-performance liquid chromatography (RP-HPLC) and solid-phase extraction (SPE) on a cation-exchange/reversed-phase STRATA-XC cartridge - were used with the purpose of confirming glycosylation profiles. Oligosaccharides were analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) after derivatization with phenylhydrazine and/or permethylation. Characteristic fragment ions produced under MS/MS conditions allowed discrimination between isomeric structures of oligosaccharides, including those sialylated with two types of acidic residues. The complementary methods allowed structural characterization of oligosaccharides from various N-glycan classes. Furthermore, to validate results, glycosylation profiles of woodchuck sera were compared to glycans obtained from mouse serum on the same conditions. In summary, we have identified 40 N-glycan structures in the serum of woodchucks and some types of oligosaccharide structures appeared to increase in HCC samples following protease digest. The study provides improved tools for the characterization of N-glycans from total serum in the progression of liver disease.

Mass spectrometric profiling of N-linked oligosaccharides and uncommon glycoform in mouse serum with head and neck tumor.

N-linked oligosaccharides obtained from total serum of mice with implanted head and neck tumors were analyzed and compared with those from control samples of healthy mice. Methods used include a combination of a derivatization procedure with phenylhydrazine (PHN) and analysis by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Oligosaccharides were enzymatically released from total serum with PNGaseF and purified by high-performance liquid chromatography (HPLC) on a reversed-phase column. Mass spectra contained ion peaks of labeled oligosaccharides and MS/MS experiments provided useful data for the structural elucidation of these compounds. More than 40 N-glycans with compositions characteristic of high-mannose, hybrid, complex, neutral, and sialylated structures were identified in the serum of tumoral mice. Significant differences between samples were observed with respect to the abundances of high mannose and hybrid glycans. These oligosaccharides showed higher relative intensities in the spectra obtained from the cancer sera. Complex sialylated oligosaccharides had similar abundances in both types of sera, with the exception of fucosylated biantennary disialylated oligosaccharide, which was mostly detected with lower abundance in control samples. In the MALDI spectra, several minor species corresponded to uncommon carbohydrates. These structures have been investigated in detail by MS/MS. Among these novel glycoforms, a few sialylated oligosaccharides without a free reducing end were identified. Also, glycans with an extra 60 u were observed and likely feature the presence of a 2-acetamido-2-deoxyoctose residue attached on antennae of 3- or 6-linked mannose.

Nonretentive solid-phase extraction of phosphorylated peptides from complex peptide mixtures for detection by matrix-assisted laser desorption/ionization mass spectrometry.

Widespread interest in protein phosphorylation has led to the development of a variety of methods for the analysis of phosphoproteomes of different types of organisms. Many applications involve pretreatment of the sample before mass spectrometric measurement and can crucially improve the detection efficiency of individual phosphopeptides. Despite intense research efforts, separation and extraction of phosphorylated peptides, especially multiphosphorylated ones, remain challenging tasks and need to be further explored and expanded with unconventional approaches. In this study, we describe the application of nonretentive solid-phase extraction (SPE) to the analysis of phosphopeptides using the highly cross-linked polystyrene-divinylbenzene material Strata-X. This study indicates that the procedure allows for the preferential extraction of phosphopeptides regardless of their extent of phosphorylation. The Strata-X material primarily retains nonphosphorylated peptides by hydrophobic interaction, whereas the inherent hydrophilicity of phosphorylated peptides leads to their partitioning into the aqueous phase. Phosphopeptides that were rapidly segregated out of tryptic digest mixtures and collected in the early aqueous fractions generated intense signals in mass spectra. The method was developed using SPE Strata-X columns, then suited for detection and sequencing of phosphopeptides by miniaturizing the system to the scale of custom-made microcolumns. This provided fast isolation of phosphopeptides from protein digests along with direct MALDI on-target deposition. The possibility of on-target washing during sample preparation is also presented.

Method for investigation of oligosaccharides from glycopeptides: direct determination of glycosylation sites in proteins.

Characterization of glycopeptides has become an important tool toward a better understanding of the molecular details in carbohydrate-protein interactions. In this approach, oligosaccharides are commonly not detectable under mass spectrometric conditions because of ionization suppression by deglycosylated peptides. Their composition is only deduced from the mass differences between glycopeptides and corresponding deglycosylated peptides. Here, we describe how carbohydrates can be easily detected in the PNGase-treated samples and structurally investigated next to the peptides. The efficacy of this method is demonstrated through the analysis of tryptic glycopeptides obtained from human IgG. Following deglycosylation with PNGaseF and derivatization with phenylhydrazine, MALDI spectra produced ion peaks of labeled oligosaccharides and deglycosylated peptides. The relative abundances of individual oligosaccharides were consistent with those of the glycopeptides. MALDI-MS/MS provided useful data for the structural elucidation of oligosaccharides, including the assignment of dominant isomers and glycosylation sites in peptides. MALDI-MS/MS fragmentation patterns of deglycosylated peptide ions indicated glycosylation sites at asparagine 297 and 299. The observed peptide of the composition ADQTVYR, described for the first time in this study, indicated new glycosylation sites in IgG1 human myeloma plasma.