Cytoplasmic Shotgun Proteomic Points to Key Proteins and Pathways in Temozolomide-Resistant Glioblastoma Multiforme.

Temozolomide (TMZ) is the first line of chemotherapy to treat primary brain tumors of the type glioblastoma multiforme (GBM). TMZ resistance (TMZR) is one of the main barriers to successful treatment and is a principal factor in relapse, resulting in a poor median survival of 15 months. The present paper focuses on proteomic analyses of cytosolic fractions from TMZ-resistant (TMZR) LN-18 cells. The experimental workflow includes an easy, cost-effective, and reproducible method to isolate subcellular fraction of cytosolic (CYTO) proteins, mitochondria, and plasma membrane proteins for proteomic studies. For this study, enriched cytoplasmic fractions were analyzed in replicates by nanoflow liquid chromatography tandem high-resolution mass spectrometry (nLC-MS/MS), and proteins identified were quantified using a label-free approach (LFQ). Statistical analysis of control (CTRL) and temozolomide-resistant (TMZR) proteomes revealed proteins that appear to be differentially controlled in the cytoplasm. The functions of these proteins are discussed as well as their roles in other cancers and TMZ resistance in GBM. Key proteins are also described through biological processes related to gene ontology (GO), molecular functions, and cellular components. For protein-protein interactions (PPI), network and pathway involvement analyses have been performed, highlighting the roles of key proteins in the TMZ resistance phenotypes. This study provides a detailed insight into methods of subcellular fractionation for proteomic analysis of TMZ-resistant GBM cells and the potential to apply this approach to future large-scale studies. Several key proteins, protein-protein interactions (PPI), and pathways have been identified, underlying the TMZ resistance phenotype and highlighting the proteins' biological functions.

Compendium of Chromatographic Behavior of Post-translationally and Chemically Modified Peptides in Bottom-Up Proteomic Experiments.

We have systematically evaluated the chromatographic behavior of post-translationally/chemically modified peptides using data spanning over 70 of the most relevant modifications. These retention properties were measured for standard bottom-up proteomic settings (fully porous C18 separation media, 0.1% formic acid as ion-pairing modifier) using collections of modified/nonmodified peptide pairs. These pairs were generated by spontaneous degradation, chemical or enzymatic treatment, analysis of synthetic peptides, or the cotranslational incorporation of noncanonical proline analogues. In addition, these measurements were validated using external data acquired for synthetic peptides and enzymatically induced citrullination. Working in units of hydrophobicity index (HI, % ACN) and evaluating the average retention shifts (ΔHI) represent the simplest approach to describe the effect of modifications from a didactic point of view. Plotting HI values for modified (y-axis) vs nonmodified (x-axis) counterparts generates unique slope and intercept values for each modification defined by the chemistry of the modifying moiety: its hydrophobicity, size, pKa of ionizable groups, and position of the altered residue. These composition-dependent correlations can be used for coarse incorporation of PTMs into models for prediction of peptide retention. More accurate predictions would require the development of specific sequence-dependent algorithms to predict ΔHI values.

Recent advancements in glycoproteomic studies: Glycopeptide enrichment and derivatization, characterization of glycosylation in SARS CoV2, and interacting glycoproteins.

Proteomics studies allow for the determination of the identity, amount, and interactions of proteins under specific conditions that allow the biological state of an organism to ultimately change. These conditions can be either beneficial or detrimental. Diseases are due to detrimental changes caused by either protein overexpression or underexpression caused by as a result of a mutation or posttranslational modifications (PTM), among other factors. Identification of disease biomarkers through proteomics can be potentially used as clinical information for diagnostics. Common biomarkers to look for include PTM. For example, aberrant glycosylation of proteins is a common marker and will be a focus of interest in this review. A common way to analyze glycoproteins is by glycoproteomics involving mass spectrometry. Due to factors such as micro- and macroheterogeneity which result in a lower abundance of each version of a glycoprotein, it is difficult to obtain meaningful results unless rigorous sample preparation procedures are in place. Microheterogeneity represents the diversity of glycans at a single site, whereas macroheterogeneity depicts glycosylation levels at each site of a protein. Enrichment and derivatization of glycopeptides help to overcome these limitations. Over the time range of 2016 to 2020, several methods have been proposed in the literature and have contributed to drastically improve the outcome of glycosylation analysis, as presented in the sampling surveyed in this review. As a current topic in 2020, glycoproteins carried by pathogens can also cause disease and this is seen with SARS CoV2, causing the COVID-19 pandemic. This review will discuss glycoproteomic studies of the spike glycoprotein and interacting proteins such as the ACE2 receptor.

Liquid chromatography-tandem mass spectrometry glycoproteomic study of porcine IgG and detection of subtypes.

RATIONALE: While high-throughput proteomic methods have been widely applied to monoclonal antibodies and human immunoglobulin gamma (IgG) samples, less information is available on porcine IgG. As pigs are considered one of the most suitable species for xenotransplantation, it is important to characterize IgG amino acid sequences and glycosylation profiles, which is the focus of this study. METHODS: Three different purified porcine IgG samples, including wild-type and knockout species, were digested with trypsin and enriched for glycopeptides. Digestion mixtures were spiked with a mixture of six standard peptides. Analysis was performed using electrospray ionization liquid chromatography-tandem mass spectrometry (MS/MS) in standard MS/MS data-dependent acquisition mode on a hybrid triple quadrupole time-of-flight mass spectrometer. RESULTS: To facilitate the classification of subtypes detected experimentally, UniprotKB database entries were organized using comparative alignment scores. Sequences were grouped based on 11 different subtypes as translated from GenBank entries. Proteomic searches were accomplished automatically using specialized software, whereas glycoprotein searches were performed manually by monitoring the extracted chromatograms of diagnostic MS/MS glycan fragments and studying their corresponding mass spectra; 40-50 non-glycosylated peptides and 4-5 glycosylated peptides were detected in each sample, with several glycoforms per sequence. CONCLUSIONS: Proteomic analysis of porcine IgG is complicated by factors such as the presence of several subtypes, redundant heavy chain (HC) sequences in protein databases, and the lack of consistent cross-referencing between databases. Aligning and comparing HC sequences were necessary to eliminate redundancy. This study highlights the complexity of pig IgG and shows the importance of MS in proteomics and glycoproteomics.

Retention Time Prediction for Glycopeptides in Reversed-Phase Chromatography for Glycoproteomic Applications.

The sequence-specific retention calculator algorithm (SSRCalc) [ Krokhin , O. V. Anal. Chem. 2006 , 78 , 7785 ] was adapted for the prediction of retention times of N-glycopeptides separated by reversed-phase high performance liquid chromatography (RPLC). The retention time shifts (dHI = HIglyco - HIdeglyco, where HI is the hydrophobicity index, measured in percent acetonitrile units) used for modeling were measured for 602 glycopeptides versus 123 of their deglycosylated analogues. Our method used a tryptic digest of 12 purified glycoproteins, glycopeptide enrichment, deglycosylation with PNGaseF, and RPLC-MS/MS analysis of combined (deglycosylated and intact) peptide mixtures. On average, glycosylation yields a 0.79% acetonitrile unit decrease in retention, compared with the hydrophobicity indices of their deglycosylated analogues. These values, however, are drastically different for asialo (-1.37% acetonitrile units), monosialylated (-0.47% acetonitrile units), disialylated (+0.61% acetonitrile units), and trisialylated (+1.94% acetonitrile units) glycans. Peptide retention time shifts upon glycosylation (dHI) vary depending on the number of monosaccharide units, the presence or absence of sialic acid, peptide hydrophobicity, and the number of position-dependent features. These features are mostly driven by competing effects of acidic residues (aspartic acid and sialic acid) on ion-pair formation and by nearest-neighbor effects of hydrophilic glycans. The accuracy of the modified prediction model for glycopeptides approaches that of the prediction for nonmodified species (R2 = 0.97 vs 0.98). However, retention time prediction based on the experimental retention values of deglycosylated analogues (HIglyco = HIdeglyco + dHI, R2 = 0.995) is much more accurate, thus providing a solid support for glycopeptide identification in complex samples based on mass and retention time.

Elicited and pre-existing anti-Neu5Gc antibodies differentially affect human endothelial cells transcriptome.

Humans cannot synthesize N-glycolylneuraminic acid (Neu5Gc) but dietary Neu5Gc can be absorbed and deposited on endothelial cells (ECs) and diet-induced anti-Neu5Gc antibodies (Abs) develop early in human life. While the interaction of Neu5Gc and diet-induced anti-Neu5Gc Abs occurs in all normal individuals, endothelium activation by elicited anti-Neu5Gc Abs following a challenge with animal-derived materials, such as following xenotransplantation, had been postulated. Ten primary human EC preparations were cultured with affinity-purified anti-Neu5Gc Abs from human sera obtained before or after exposure to Neu5Gc-glycosylated rabbit IgGs (elicited Abs). RNAs of each EC preparation stimulated in various conditions by purified Abs were exhaustively sequenced. EC transcriptomic patterns induced by elicited anti-Neu5Gc Abs, compared with pre-existing ones, were analyzed. qPCR, cytokines/chemokines release, and apoptosis were tested on some EC preparations. The data showed that anti-Neu5Gc Abs induced 967 differentially expressed (DE) genes. Most DE genes are shared following EC activation by pre-existing or anti-human T-cell globulin (ATG)-elicited anti-Neu5Gc Abs. Compared with pre-existing anti-Neu5Gc Abs, which are normal component of ECs environment, elicited anti-Neu5Gc Abs down-regulated 66 genes, including master genes of EC function. Furthermore, elicited anti-Neu5Gc Abs combined with complement-containing serum down-regulated most transcripts mobilized by serum alone. Both types of anti-Neu5Gc Abs-induced a dose- and complement-dependent release of selected cytokines and chemokines. Altogether, these data show that, compared with pre-existing anti-Neu5Gc Abs, ATG-elicited anti-Neu5Gc Abs specifically modulate genes related to cytokine responses, MAPkinase cascades, chemotaxis, and integrins and do not skew the EC transcriptome toward a pro-inflammatory profile in vitro.

Characterization of immunoglobulins through analysis of N-glycopeptides by MALDI-TOF MS.

The aim of this report is to emphasize the role, usefulness and power of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) in the analysis of glycoforms of antibodies (Abs) through their proteolytic glycopeptides. Abs are complex biomolecules in which glycans hold determinant properties and thus need to be thoroughly characterized following Ab production by recombinant methods or Ab collection from human/animal serum or tissue. In spite of the great robustness of MALDI-TOF MS in terms of tolerance to impurities, the analysis of Abs and Ab components using this technique requires extensive sample preparation involving all or some of chromatography, solid phase extraction, enzymatic modification, and chemical derivatization. This report focuses on a monoclonal Ab produced in cell culture, as well as on a polyclonal human immunoglobulin (Ig) G obtained commercially and a polyclonal porcine IgG obtained from serum. A method is first provided to separate Ab protein chain components (light chains, heavy chains) by gel electrophoresis, which is useful for instance for protein-A eluates of Igs either from cell culture or biological samples. This allows for in-gel proteolytic digestion of the protein gel band(s) of choice for further MS characterization. Also discussed is the more conventional in-solution overnight digestion method used here with each of two proteolytic enzymes, i.e. trypsin and chymotrypsin. The overnight method is in turn compared with a much faster approach, that of digesting Abs with trypsin or chymotrypsin through the action of microwave heating. For method comparison, glycopeptides are fractionated from digestion mixtures using mostly C-18 cartridges for simplicity, although this enrichment procedure is also compared with other published procedures. The advantages of MALDI tandem mass spectrometry are highlighted for glycopeptide analysis, and lastly an esterification method applied to glycopeptides is discussed for retention of sialic acid residues on peptide acidic glycoforms.

Rapid screening of Alternaria mycotoxins using MALDI-TOF mass spectrometry.

BACKGROUND: Members of the Alternaria genus produce various toxins whose occurrence in agricultural commodities is a major concern for humans and the environment. The present study developed a simple and efficient matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) method for the rapid detection of Alternaria toxins. RESULTS: A new method for the detection of alternariol (AOH), alternariol monomethyl ether (AME) and tentoxin (TEN) by MALDI-TOF MS was developed. Different solid phase extraction (SPE) clean-up methods were tried to optimize the purification of wheat matrix, and an optimal extraction method was designed to recover the three Alternaria toxins. In addition, various MALDI matrices were examined and α-cyano-4-hydroxycinnamic acid (CHCA) matrix gave good repeatability for all three Alternaria toxins. CONCLUSION: This is the first study to report the detection of three important Alternaria toxins concurrently using MALDI-TOF MS and opens up the possibility of rapid screening of Alternaria toxins in several other cereals and food products. © 2016 Her Majesty the Queen in Right of Canada Journal of the Science of Food and Agriculture © 2016 Society of Chemical Industry.

Characterization of N-glycosylation and amino acid sequence features of immunoglobulins from swine.

The primary goal of this study was to develop a method to study the N-glycosylation of IgG from swine in order to detect epitopes containing N-glycolylneuraminic acid (Neu5Gc) and/or terminal galactose residues linked in α1-3 susceptible to cause xenograft-related problems. Samples of immunoglobulin were isolated from porcine serum using protein-A affinity chromatography. The eluate was then separated on electrophoretic gel, and bands corresponding to the N-glycosylated heavy chains were cut off the gel and subjected to tryptic digestion. Peptides and glycopeptides were separated by reversed phase liquid chromatography and fractions were collected for matrix-assisted laser desorption/ionization time-of-flight mass spectrometric (MALDI-TOF-MS) analysis. Overall no α1-3 galactose was detected, as demonstrated by complete susceptibility of terminal galactose residues to ß-galactosidase digestion. Neu5Gc was detected on singly sialylated structures. Two major N-glycopeptides were found, EEQFNSTYR and EAQFNSTYR as determined by tandem MS (MS/MS), as previously reported by Butler et al. (Immunogenetics, 61, 2009, 209-230), who found 11 subclasses for porcine IgG. Out of the 11, ten include the sequence corresponding to EEQFNSTYR, and only one codes for EAQFNSTYR. In this study, glycosylation patterns associated with both chains were slightly different, in that EEQFNSTYR had a higher content of galactose. The last step of this study consisted of peptide-mapping the 11 reported porcine IgG sequences. Although there was considerable overlap, at least one unique tryptic peptide was found per IgG sequence. The workflow presented in this manuscript constitutes the first study to use MALDI-TOF-MS in the investigation of porcine IgG structural features.

Multiplexed azido-group isotopic capture (MAGIC) beads: Selective analysis of azido compounds using a propargyl-based cleavable linker, a proof of concept.

RATIONALE: A cleavable linker is designed and synthesized for the selective capture of azide-containing compounds. This article presents a proof of concept methodology involving the use of peptide-functionalized aminopropyl silica, on which the peptide is constructed by solid-phase peptide synthesis. METHODS: The peptide linker has L-propargylglycine (Pra) at one terminal end to allow the conjugation of azide-containing molecules by copper assisted azide alkyne cycloaddition, also known as click reaction. L-Arginine (Arg) is placed just before Pra to permit the release of the captured product by tryptic cleavage. Three glycine (Gly) residues, as part of the linker, are appended to the silica bead to present a spacer section that allows efficient tryptic cleavage devoid of steric hindrance imposed by the bulky bead. The bead composition is Si-O-propyl-NH-Gly-Gly-Gly-Arg-Pra. RESULTS: This solid-phase material can be used to capture and release azide-functionalized compounds. The beads are first tested on three azido compounds, 2-azido-2-deoxyglucose (ADG), BOC-p-azido-Phe-OH (BAzPhe), where BOC = tert-butoxycarbonyl, and tetraacetylated-N-azidomannosamine (Ac4 ManNAz). Copper-mediated click reaction conditions are used and released products are characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and tandem MS (MS/MS). CONCLUSIONS: This method allows easy identification of captured compounds based on mass and fragmentation analysis. Moreover, it is useful for the analysis of small azide-containing compounds by MALDI-TOF-MS which may not be possible otherwise due to matrix interferences. The insertion of isotopically labeled Arg residues provides the possibility of multiplex analysis, from which the beads have been called MAGIC (for Multiplexed Azido-Group Isotopic Capture). Copyright © 2016 John Wiley & Sons, Ltd.

Algal carbohydrates affect polyketide synthesis of the lichen-forming fungus Cladonia rangiferina.

Lichen secondary metabolites (polyketides) are produced by the fungal partner, but the role of algal carbohydrates in polyketide biosynthesis is not clear. This study examined whether the type and concentration of algal carbohydrate explained differences in polyketide production and gene transcription by a lichen fungus (Cladonia rangiferina). The carbohydrates identified from a free-living cyanobacterium (Spirulina platensis; glucose), a lichen-forming alga (Diplosphaera chodatii; sorbitol) and the lichen alga that associates with C. rangiferina (Asterochloris sp.; ribitol) were used in each of 1%, 5% and 10% concentrations to enrich malt yeast extract media for culturing the mycobiont. Polyketides were determined by high performance liquid chromatography (HPLC), and polyketide synthase (PKS) gene transcription was measured by quantitative PCR of the ketosynthase domain of four PKS genes. The lower concentrations of carbohydrates induced the PKS gene expression where ribitol up-regulated CrPKS1 and CrPKS16 gene transcription and sorbitol up-regulated CrPKS3 and CrPKS7 gene transcription. The HPLC results revealed that lower concentrations of carbon sources increased polyketide production for three carbohydrates. One polyketide from the natural lichen thallus (fumarprotocetraric acid) also was produced by the fungal culture in ribitol supplemented media only. This study provides a better understanding of the role of the type and concentration of the carbon source in fungal polyketide biosynthesis in the lichen Cladonia rangiferina.

A systematic study of glycopeptide esterification for the semi-quantitative determination of sialylation in antibodies.

RATIONALE: In the expression of recombinant proteins, an important parameter to control or influence is their level of sialylation. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometric (MS) methods tend to either underestimate (positive mode) or overestimate (negative mode) the content of sialylated vs. neutral glycans in glycoproteins. Esterification methods have been developed for free sialylated glycans and sialylated Asn-glycans, allowing these acidic groups to ionize with the same efficiency as neutral sugars. METHODS: Here we describe a method which modifies glycopeptides by esterification. This simple procedure is applied to glycopeptides isolated from tryptic digests of monoclonal antibodies (mAbs), some highly sialylated. To better understand the effect of esterification on the peptide backbone, synthetic EEQYNSTYR was esterified and studied by tandem mass spectrometry (MS/MS). Acetamidation of EEQYNSTYR was also studied as some mAb samples had been overalkylated prior to tryptic digestion. RESULTS: As a general trend, ethyl-esterification or lactonization is observed for each sialic acid on glycoforms of EEQYNSTYR (the N-glycosylated tryptic peptide of IgG Fc), depending on the branching position of the sialic acid (α2,3 or α2,6). Esterification also affects the carboxyl groups in the peptide, including the C-terminal COOH. CONCLUSIONS: For antibody analysis, MALDI-MS ion abundances give a better semi-quantitative estimate of sialylation levels for esterified than for unreacted glycopeptides. The method is simple to use and helps to differentiate the branching patterns of sialic acids in antibodies.

Mass spectrometric analysis of products of metabolic glycan engineering with azido-modification of sialic acids.

Metabolic engineering of glycans present on antibodies and other glycoproteins is becoming an interesting research area for improving our understanding of the glycome. With knowledge of the sialic acid biosynthetic pathways, the experiments described in this report are based on a published procedure involving the addition of a synthesized azido-mannosamine sugar into cell culture media and evaluation of downstream expression as azido-sialic acid. This unique bioorthogonal sugar has the potential for a variety of "click chemistry" reactions through the azide linkage, which allow for it to be isolated and quantified given the choice of label. In this report, mass spectrometry was used to investigate and optimize the cellular absorption of peracetylated N-azidoacetylmannosamine (Ac4ManNAz) to form N-azidoacetylneuraminic acid (SiaNAz) in a Chinese hamster ovary (CHO) cell line transiently expressing a double mutant trastuzumab (TZMm2), human galactosyltransferase 1 (GT), and human α-2,6-sialyltransferase (ST6). This in vivo approach is compared to in vitro enzymatic addition SiaNAz onto TZMm2 using soluble ß-galactosamide α-2,6-sialyltransferase 1 and CMP-SiaNAz as donor. The in vivo results suggest that for this mAb, concentrations above 100 µM of Ac4ManNAz are necessary to allow for observation of terminal SiaNAz on tryptic peptides of TZMm2 by matrix-assisted laser desorption ionization (MALDI) mass spectrometry. This is further confirmed by a parallel study on the production of EG2-hFc monoclonal antibody (Zhang J et al. Prot Expr Purific 65(1); 77-82, 2009) in the presence of increasing concentrations of Ac4ManNAz.

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.

Peptide retention time prediction for electrostatic repulsion-hydrophilic interaction chromatography.

Electrostatic Repulsion-Hydrophilic Interaction Chromatography (ERLIC) is one of the legacy separation tools developed by Dr. Andrew Alpert and has been used for developing unique separation methods of hydrophilic compounds, including peptides. In the past it has been studied using designed peptide libraries to elucidate major features of its separation mechanism, while comprehensive peptide retention modeling for ERLIC is still lacking. In this work we employed a proteomics-derived ∼170,000 peptide retention datasets to evaluate major ERLIC retention features using the framework of our Sequence-Specific Retention Calculator model. The separation conditions were adjusted to obtain a wider proteome coverage, particularly for non-modified peptides, resulting in a superior separation orthogonality for a 2D LC combination with reversed-phase C18 LC-MS in the second dimension. The SSRCalc ERLIC model presents a consistent theme with the existing ERLIC retention mechanism, reflecting a dependence on peptide orientation and the position of charged and hydrophilic residues across the peptide backbone. R2 values of 0.935 and 0.955 accuracy were demonstrated for the standard interpretable SSRCalc model and machine learning algorithm, respectively. The effects of various PTMs on peptide retention were evaluated in this study, covering spontaneous (oxidation, deamidation) and enzymatic (N-terminal acetylation, phosphorylation, glycosylation) modifications.

Qualitative and quantitative assessment on the use of magnetic nanoparticles for glycopeptide enrichment.

Glycoproteomics represent the field of study of the dynamic changes occurring among glycoconjugates within the cellular compartments. Changes in glycosylation have been linked to various diseases, including metastatic carcinomas in which the 9 carbon sialic acid moiety has been shown to play a prominent role. The common method used to study these aberrant changes most often includes a mass spectrometer at some stage in the workflow. However, serum samples contain many proteins which inhibit the analysis of these glycosylation changes, and ergo, enrichment steps are employed as a measure to help alleviate this ailment. Routinely, this is accomplished using lectins, either alone or in combination, to retrieve proteins with specific sugar linkages within the serum sample. This methodology, although known to be very specific, requires many washing steps, making it a cumbersome addition to a high throughput workflow. Presented here is an alternative protocol using custom-made amine functionalized magnetic nanoparticles (MNP) which are nearly 4× smaller than those used before for similar purposes. The developed protocol is based on both hydrophilic interaction and weak anion exchange principles, allowing it to target glycopeptides but, more specifically, those which contain sialylation. For quantification purposes, tandem mass tags from Thermo Scientific were utilized to compare the enrichment efficiencies between the magnetic nanoparticle method and a commercially available glycopeptide enrichment kit offered through EMD Millipore. The MNP method is fast (~10 min) and simple and can quantitatively and qualitatively enrich sialylated glycopeptides more than the commercially available kit.

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.

An overview of glioblastoma multiforme and temozolomide resistance: can LC-MS-based proteomics reveal the fundamental mechanism of temozolomide resistance?

Glioblastoma multiforme (GBM) is a primary type of lethal brain tumor. Over the last two decades, temozolomide (TMZ) has remained the primary chemotherapy for GBM. However, TMZ resistance in GBM constitutes an underlying factor contributing to high rates of mortality. Despite intense efforts to understand the mechanisms of therapeutic resistance, there is currently a poor understanding of the molecular processes of drug resistance. For TMZ, several mechanisms linked to therapeutic resistance have been proposed. In the past decade, significant progress in the field of mass spectrometry-based proteomics has been made. This review article discusses the molecular drivers of GBM, within the context of TMZ resistance with a particular emphasis on the potential benefits and insights of using global proteomic techniques.

Challenges and opportunities for proteomics and the improvement of bread wheat quality.

Wheat remains a critical global food source, pressured by climate change and the need to maximize yield, improve processing and nutritional quality and ensure safety. An enormous amount of research has been conducted to understand gluten protein composition and structure in relation to end-use quality, yet progress has become stagnant. This is mainly due to the need and inability to biochemically characterize the intact functional glutenin polymer in order to correlate to quality, necessitating reduction to monomeric subunits and a loss of contextual information. While some individual gluten proteins might have a positive or negative influence on gluten quality, it is the sum total of these proteins, their relative and absolute expression, their sub-cellular trafficking, the amount and size of glutenin polymers, and ratios between gluten protein classes that define viscoelasticity of gluten. The sub-cellular trafficking of gluten proteins during seed maturation is still not completely clear and there is evidence of dual pathways and therefore different destinations for proteins, either constitutively or temporally. The trafficking of proteins is also unclear in endosperm cells as they undergo programmed cell death; Golgi disappear around 12 DPA but protein filling continues at least to 25 DPA. Modulation of the timing of cellular events will invariably affect protein deposition and therefore gluten strength and function. Existing and emerging proteomics technologies such as proteoform profiling and top-down proteomics offer new tools to study gluten protein composition as a whole system and identify compositional patterns that can modify gluten structure with improved functionality.

Increased phosphorylation of HexM improves lysosomal uptake and potential for managing GM2 gangliosidoses.

Tay-Sachs and Sandhoff diseases are genetic disorders resulting from mutations in HEXA or HEXB, which code for the α- and ß-subunits of the heterodimer ß-hexosaminidase A (HexA), respectively. Loss of HexA activity results in the accumulation of GM2 ganglioside (GM2) in neuronal lysosomes, culminating in neurodegeneration and death, often by age 4. Previously, we combined critical features of the α- and ß-subunits of HexA into a single subunit to create a homodimeric enzyme known as HexM. HexM is twice as active as HexA and degrades GM2 in vivo, making it a candidate for enzyme replacement therapy (ERT). Here we show HexM production is scalable to meet ERT requirements and we describe an approach that enhances its cellular uptake via co-expression with an engineered GlcNAc-1-phosphotransferase that highly phosphorylates lysosomal proteins. Further, we developed a HexA overexpression system and functionally compared the recombinant enzyme to HexM, revealing the kinetic differences between the enzymes. This study further advances HexM as an ERT candidate and provides a convenient system to produce HexA for comparative studies.