Site-Specific Glycan Microheterogeneity Evaluation of Aflibercept Fusion Protein by Glycopeptide-Based LC-MSMS Mapping.

The evaluation of the protein glycosylation states of samples of aflibercept obtained from three different regions was conducted by site-specific N-linked glycan microheterogeneity profiling. Glycopeptide-based nano-LC MSMS mapping of tryptic-digested samples of each aflibercept lot provided site-specific information about glycan microheterogeneity on each of the five N-glycosylation sites (two sites in the VEGFR-1 region, two sites in the VEGFR-2 region, and one site in the human IgG Fc region). Next, the glycopeptide-mapping results obtained from the three different aflibercept lots were compared to evaluate the similarity between the samples.

Compositional Characteristics of Glucuronide Conjugates in Regional var. Herbal Extracts Using a Set of Polyphenolic Marker Compounds.

var. (GHL) is one of many herbal plants widely used in hot herbal teas and in oriental prescriptions to treat various diseases. Although the beneficial effects of GHL may be influenced by differences in the composition of active constituents in the herbal extracts, there are few reports on the compositional characteristics of GHL herbal extracts to date.

Identification of Polyphenol Glucuronide Conjugates in var. Hot Water Extracts by High-Performance Liquid Chromatography-Tandem Mass Spectrometry (HPLC-MS/MS).

var. (GHL) is one of many herbal plants distributed worldwide and is known to contain various biologically useful antioxidant constituents. GHL has been used in folk remedies for various treatments and as favorable tea beverages.

Investigation of Site-Specific Differences in Glycan Microheterogeneity by N-Glycopeptide Mapping of VEGFR-IgG Fusion Protein.

A biosimilar fusion protein VEGFR-IgG consisting of vascular endothelial growth factor receptors 1 and 2 (VEGFR-1, VEGFR-2) and the Fc portion of human IgG1 was prepared for this study. The prepared fusion protein was expected to possess a total of five N-linked glycosylation sites: two sites in the VEGFR-1 region, two sites in the VEGFR-2 region, and one site in the human IgG Fc region. For site-specific glycan analysis, the fusion protein was hydrolyzed with trypsin, and the resulting tryptic digests were analyzed by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI MS/MS).

Comparative Glycopeptide Analysis for Protein Glycosylation by Liquid Chromatography and Tandem Mass Spectrometry: Variation in Glycosylation Patterns of Site-Directed Mutagenized Glycoprotein.

Glycosylation is one of the most important posttranslational modifications for proteins, including therapeutic antibodies, and greatly influences protein physiochemical properties. In this study, glycopeptide mapping of a reference and biosimilar recombinant antibodies (rAbs) was performed using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) and an automated Glycoproteome Analyzer (GPA) algorithm. The tandem mass analyses for the reference and biosimilar samples indicate that this approach proves to be highly efficient in reproducing consistent analytical results and discovering the implications of different rAb production methods on glycosylation patterns.

Analysis of fucosylation in liver-secreted N-glycoproteins from human hepatocellular carcinoma plasma using liquid chromatography with tandem mass spectrometry.

Fucosylation of N-glycoproteins has been implicated in various diseases, such as hepatocellular carcinoma (HCC). However, few studies have performed site-specific analysis of fucosylation in liver-secreted proteins. In this study, we characterized the fucosylation patterns of liver-secreted proteins in HCC plasma using a workflow to identify site-specific N-glycoproteins, where characteristic B- and/or Y-ion series with and without fucose in collision-induced dissociation were used in tandem mass spectrometry.

MALDI MS-based Composition Analysis of the Polymerization Reaction of Toluene Diisocyanate (TDI) and Ethylene Glycol (EG).

This study describes an MS-based analysis method for monitoring changes in polymer composition during the polyaddition polymerization reaction of toluene diisocyanate (TDI) and ethylene glycol (EG). The polymerization was monitored as a function of reaction time using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS). The resulting series of polymer adducts terminated with various end-functional groups were precisely identified and the relative compositions of those series were estimated.

Quantitative analysis of low-abundance serological proteins with peptide affinity-based enrichment and pseudo-multiple reaction monitoring by hybrid quadrupole time-of-flight mass spectrometry.

Multiple reaction monitoring (MRM) is commonly used for the quantitative analysis of proteins during mass pectrometry (MS), and has excellent specificity and sensitivity for an analyte in a complex sample. In this study, a pseudo-MRM method for the quantitative analysis of low-abundance serological proteins was developed using hybrid quadrupole time-of-flight (hybrid Q-TOF) MS and peptide affinity-based enrichment. First, a pseudo-MRM-based analysis using hybrid Q-TOF MS was performed for synthetic peptides selected as targets and spiked into tryptic digests of human serum.

Quantitative mass spectrometric analysis of glycoproteins combined with enrichment methods.

Mass spectrometry (MS) has been a core technology for high sensitive and high-throughput analysis of the enriched glycoproteome in aspects of quantitative assays as well as qualitative profiling of glycoproteins. Because it has been widely recognized that aberrant glycosylation in a glycoprotein may involve in progression of a certain disease, the development of efficient analysis tool for the aberrant glycoproteins is very important for deep understanding about pathological function of the glycoprotein and new biomarker development. This review first describes the protein glycosylation-targeting enrichment technologies mainly employing solid-phase extraction methods such as hydrizide-capturing, lectin-specific capturing, and affinity separation techniques based on porous graphitized carbon, hydrophilic interaction chromatography, or immobilized boronic acid.

Differential proteomic approach for identification and verification of aberrantly glycosylated proteins in adenocarcinoma lung cancer (ADLC) plasmas by lectin-capturing and targeted mass spectrometry.

Unlabelled: To investigate quantitative differences in aberrant glycosylation of target glycoproteins between noncancerous group and patient group with adenocarcinoma lung cancer (ADLC), differential proteomic approach was developed by cooperatively using comparative lectin-capturing, targeted mass spectrometry (MRM MS), and antibody/lectin sandwich ELISA. Plasma samples comparatively prepared from 3 ADLC patients and 3 controls, with and without lectin-fractionation using fucose-specific Aleuria aurantia lectin (AAL), were trypsin-digested and analyzed for target glycoproteins, alpha-1-acid glycoprotein (AGP) and ceruloplasmin (CP), by MRM MS. From the MRM MS data the abundance levels of AAL-captured glycoforms of both targets were significantly higher in ADLC cases compared to controls, although the levels in total protein abundance were comparable between ADLC and control groups.

MRM validation of targeted nonglycosylated peptides from N-glycoprotein biomarkers using direct trypsin digestion of undepleted human plasma.

Unlabelled: A rapid, simple, and reproducible MRM-based validation method for serological glycoprotein biomarkers in clinical use was developed by targeting the nonglycosylated tryptic peptides adjacent to N-glycosylation sites. Since changes in protein glycosylation are known to be associated with a variety of diseases, glycoproteins have been major targets in biomarker discovery. We previously found that nonglycosylated tryptic peptides adjacent to N-glycosylation sites differed in concentration between normal and hepatocellular carcinoma (HCC) plasma due to differences in steric hindrance of the glycan moiety in N-glycoproteins to tryptic digestion (Lee et al.

Quantitative analysis of aberrant protein glycosylation in liver cancer plasma by AAL-enrichment and MRM mass spectrometry.

A lectin-coupled mass spectrometry (MS) approach was employed to quantitatively monitor aberrant protein glycosylation in liver cancer plasma. To do this, we compared the difference in the total protein abundance of a target glycoprotein between hepatocellular carcinoma (HCC) plasmas and hepatitis B virus (HBV) plasmas, as well as the difference in lectin-specific protein glycoform abundance of the target glycoprotein. Capturing the lectin-specific protein glycoforms from a plasma sample was accomplished by using a fucose-specific aleuria aurantia lectin (AAL) immobilized onto magnetic beads via a biotin-streptavidin conjugate.

Reaction monitoring of toluenediisocyanate (TDI) polymerization on a non-mixable aqueous surface by MALDI mass spectrometry.

The polymerization reaction of toluene diisocyanate (TDI) and hydroxyl compounds has been widely used for the production of polyurea resins. Since the composition and molecular-weight distribution of polymer adducts greatly influence the final properties of the resuting polymer, the development of analytical tools capable of monitoring the polyaddition reactions is important to control them as well as the properties of the resuting polymer. Here we report that matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) is useful to precisely monitor time-dependent dynamic events occurring in the polymerization reaction of TDI with water.

Overexpression and β-1,6-N-acetylglucosaminylation-initiated aberrant glycosylation of TIMP-1: a "double whammy" strategy in colon cancer progression.

There has been ongoing debate over whether tissue inhibitor of metalloproteinase-1 (TIMP-1) is pro- or anti-oncogenic. We confirmed that TIMP-1 reinforced cell proliferation in an αvβ3 integrin-dependent manner and conferred resistance against cytotoxicity triggered by TNF-α and IL-2 in WiDr colon cancer cells. The cell-proliferative effects of TIMP-1 contributed to clonogenicity and tumor growth during the onset and early phase of tumor formation in vivo and in vitro.

A lectin-coupled, targeted proteomic mass spectrometry (MRM MS) platform for identification of multiple liver cancer biomarkers in human plasma.

Aberrantly glycosylated proteins related to liver cancer progression were captured with specific lectin and identified from human plasma by multiple reaction monitoring (MRM) mass spectrometry as multiple biomarkers for hepatocellular carcinoma (HCC). The lectin fractionation for fucosylated protein glycoforms in human plasma was conducted with a fucose-specific aleuria aurantia lectin (AAL). Following tryptic digestion of the lectin-captured fraction, plasma samples from 30 control cases (including 10 healthy, 10 hepatitis B virus [HBV], and 10 cirrhosis cases) and 10 HCC cases were quantitatively analyzed by MRM to identify which glycoproteins are viable HCC biomarkers.

A lectin-coupled, multiple reaction monitoring based quantitative analysis of human plasma glycoproteins by mass spectrometry.

Aberrant protein glycosylation may be closely associated with cancer pathology. To measure the abundance of protein glycoforms with a specific glycan structure in plasma samples, we developed a lectin-coupled multiple reaction monitoring (MRM)-based mass spectrometric method. It was confirmed that the method could provide reproducible results with precision sufficient to distinguish differences in the abundance of protein glycoforms between individuals.

Identification of low-abundance cancer biomarker candidate TIMP1 from serum with lectin fractionation and peptide affinity enrichment by ultrahigh-resolution mass spectrometry.

As investigating a proteolytic target peptide originating from the tissue inhibitor of metalloproteinase 1 (TIMP1) known to be aberrantly glycosylated in patients with colorectal cancer (CRC), we first confirmed that TIMP1 is to be a CRC biomarker candidate in human serum. For this, we utilized matrix-assisted laser desorption/ionization (MALDI) Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS) showing ultrahigh-resolution and high mass accuracy. This investigation used phytohemagglutinin-L(4) (L-PHA) lectin, which shows binding affinity to the β-1,6-N-acetylglucosamine moiety of N-linked glycan on a protein, to compare fractionated aberrant protein glycoforms from both noncancerous control and CRC serum.

A multiplex lectin-channel monitoring method for human serum glycoproteins by quantitative mass spectrometry.

A mass profiling method and multiple reaction monitoring (MRM)-based quantitative approach were used to analyze multiple lectin-captured fractions of human serum using different lectins such as aleuria aurantia lectin (AAL), phytohemagglutinin-L(4) (L-PHA), concanavalin A (Con A), and Datura stramonium agglutinin (DSA) to quantitatively monitor protein glycosylation diversity. Each fraction, prepared by multiple lectin-fractionation and tryptic digestion, was analyzed by 1-D LC-MS/MS. Semi-quantitative profiling showed that the list of glycoproteins identified from each lectin-captured fraction is significantly different according to the used lectin.

Targeted mass spectrometric approach for biomarker discovery and validation with nonglycosylated tryptic peptides from N-linked glycoproteins in human plasma.

A simple mass spectrometric approach for the discovery and validation of biomarkers in human plasma was developed by targeting nonglycosylated tryptic peptides adjacent to glycosylation sites in an N-linked glycoprotein, one of the most important biomarkers for early detection, prognoses, and disease therapies. The discovery and validation of novel biomarkers requires complex sample pretreatment steps, such as depletion of highly abundant proteins, enrichment of desired proteins, or the development of new antibodies. The current study exploited the steric hindrance of glycan units in N-linked glycoproteins, which significantly affects the efficiency of proteolytic digestion if an enzymatically active amino acid is adjacent to the N-linked glycosylation site.

Galectin-3 binding protein promotes cell motility in colon cancer by stimulating the shedding of protein tyrosine phosphatase kappa by proprotein convertase 5.

It has previously been reported that shedding of the PTPκ ectodomain drives enhanced motility of colon cancer cells. Herein, we provide mechanism underlying the regulation of PTPκ shedding by galectin-3 binding protein. PTPκ was inarguably scissored by the processed form of proprotein convertase 5 (subtilisin/kexin type 5), and galectin-3 binding protein which is over-produced in colon cancer cells and tissues contributed to increased cancer cell motility by acting as a negative regulator of galectin-3 at the cell surface.

Analysis of nuclear high mobility group box 1 (HMGB1)-binding proteins in colon cancer cells: clustering with proteins involved in secretion and extranuclear function.

HMGB1 is a nuclear protein that is overexpressed and secreted in cancer cells. However, little is known about the roles of HMGB1 in the cytoplasm and secretory pathway in cancer cells. To clarify this aspect of HMGB1 function, we fractionated the cytoplasm of HCT116 colon cancer cells and used a proteomic approach to analyze cytoplasmic HMGB1-binding proteins.

Comparative quantitation of aberrant glycoforms by lectin-based glycoprotein enrichment coupled with multiple-reaction monitoring mass spectrometry.

Lectin enrichment-coupled multiple-reaction monitoring (MRM) mass spectrometry was employed to quantitatively monitor the variation of aberrant glycoforms produced under pathological states. For this, aberrant glycoforms of the tissue inhibitor of metalloproteinase 1 (TIMP1) and protein tyrosine phosphatase kappa (PTPkappa), previously known target proteins for N-acetylglucosaminyltransferase-V (GnT-V), were enriched by phytohemagglutinin-L(4) (L-PHA) lectin and comparatively analyzed in the conditioned medium of the WiDr colon cancer cell line and its GnT-V-overexpressing transfectant cells. Enriched glycoforms were digested, and the resultant peptides were comparatively quantified by MRM analysis.

Quantitative analysis of an aberrant glycoform of TIMP1 from colon cancer serum by L-PHA-enrichment and SISCAPA with MRM mass spectrometry.

Variations in glycosylation levels or in the glycoprofile of a certain glycoprotein in tumor-related sera have been widely reported and can be used as a means of differentiation. However, quantitative mass analysis of glycoproteins is difficult because of their high structural complexity and low mass sensitivity of glycopeptides. Therefore, more powerful technologies are required for the discovery of these potential biomarkers.

Non-histone nuclear factor HMGB1 is phosphorylated and secreted in colon cancers.

The high mobility group box 1 (HMGB1) protein, a non-histone nuclear factor, is overexpressed and localizes to the cytoplasm in some cancer cells. However, the mechanism of cytoplasmic HMGB1 transport, extracellular secretion, and its role in cancer progression is not clear. To simulate the activated state of HMGB1, we mutated serine residues of nuclear localization signals (NLSs) to glutamic acid and performed transfection assays.