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. Three aflibercept lots showed a high degree of similarity in glycan composition, fucosylation level, sialylation level, and branching, when all five N-glycosylation sites were assessed together as a group. On the other hand, noticeable variations between lots in the glycan types and sialylation levels on the two sites of the VEGFR-2 domain were observed when each of the five N-glycosylation sites were assessed using the glycopeptide-based method. The presence of N-glycolylneuraminic acid (NeuGc) glycans, which may mediate adverse immune reactions in antibody therapeutics, were also detected on the sites of VEGFR1 and VEGFR2 domains, but not on the IgG Fc domain site. These results imply that analyses of the glycosylation profiles of fusion proteins containing multiple N-glycosylation sites, such as aflibercept, being done as a part of quality control for the therapeutics manufacturing process or for biosimilar development, can be done with a more applicable outcome by assessing each site separately.

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

Glechoma hederacea var. longituba (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. However, research on the precise analysis of ingredients in GHL extracts remains insufficient. In this study, compositional analysis has been conducted on polyphenolic ingredients in GHL hot water extracts. GHL samples collected from growing regions were incubated in hot water at 100 °C for 1 h. The polyphenolic constituents in the hot water extracts were analyzed using high performance liquid chromatography-high resolution mass spectrometry (HPLC-HR MS) and tandem mass spectrometry (HPLC-MS/MS) in negative ion mode. As a result, a total of seven compounds were identified as the major polyphenolic constituents. Interestingly, four constituents out of the identified substances were confirmed to be polyphenol glucuronide conjugates, in which glucuronidation was known to be an important metabolic process in polyphenol aglycone along with methylation and sulphation. This study can be applied for the quality control and standardization of GHL herbal samples and the monitoring of metabolic processes involved in the polyphenolic conjugates.

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). The expected N-linked glycosylation sites were successfully identified and site-specific glycopeptide mapping was completed by Integrated GlycoProteome Analyzer (I-GPA) for the resulting raw tandem mass data. Finally, it was clearly confirmed that N-linked glycans for each glycosylation site showed significantly different patterns in microheterogeneity, which may indicate certain functions for each glycosylation site in the protein. Based on the mapping results, the unique features in glycan microheterogeneity for the five glycosylation sites of VEGFR-IgG fusion protein were compared site-specifically and further discussed to understand the functional meaning of each glycosylation pattern.

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. Second, MS-based quantitative analysis strategies coupled with the protein glycosylation-targeting enrichment technologies, by using a label-free MS, stable isotope-labeling, or targeted multiple reaction monitoring (MRM) MS, are summarized with recent published studies.

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. In total, 71 fucosylated N-glycopeptides from 13 major liver-secreted proteins in human plasma were globally identified by LC-MS/MS. Additionally, 37 fucosylated N-glycopeptides were newly identified from nine liver-secreted proteins, including alpha-1-antichymotrypsin, alpha-1-antitrypsin, alpha-2-HS-glycoprotein, ceruloplasmin, alpha-1-acid glycoprotein 1/2, alpha-2-macroglobulin, serotransferrin, and beta-2-glycoprotein 1. Of the fucosylated N-glycopeptides, bi- and tri-antennary glycoforms were the most common ones identified in liver-secreted proteins from HCC plasma. Therefore, we suggest that this analytical method is effective for characterizing fucosylation in liver-secreted proteins. Graphical abstract A global map of fucosylated and non-fucosylated glycopeptides from 13 liver-secreted glycoproteins in hepatocellular carcinoma plasma.

Overexpression and ß-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 αvß3 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. However, mass-produced TIMP-1 impeded further tumor growth by tightly inhibiting the activities of collagenases, which are critical for tumor growth and malignant transformation. Tumor cells could overcome this impasse by overexpression of N-acetylglucosaminyltransferase V, which deteriorates TIMP-1 into an aberrant glycoform. The aberrant glycoform of TIMP-1 was responsible for the mitigated inhibition of collagenases. The outbalanced activities of collagenases can degrade the basement membrane and the interstitial matrix, which act as a physical barrier for tumor growth and progression more efficiently. The concomitant overexpression of TIMP-1 and N-acetylglucosaminyltransferase V enabled WiDr cells to show a higher tumor growth rate as well as more malignant behaviors in a three-dimensional culture system.

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. Following tryptic digestion of both the total plasma and its AAL-captured fraction of each HCC and HBV sample, targeted proteomic mass spectrometry was conducted quantitatively by a multiple reaction monitoring (MRM) technique. From the MRM-based analysis of the total plasmas and AAL-captured fractions, differences between HCC and HBV plasma groups in fucosylated glycoform levels of target glycoproteins were confirmed to arise from both the change in the total protein abundance of the target proteins and the change incurred by aberrant fucosylation on target glycoproteins in HCC plasma, even when no significant change occurs in the total protein abundance level. Combining the MRM-based analysis method with the lectin-capturing technique proved to be a successful means of quantitatively investigating aberrant protein glycosylation in cancer plasma samples. Additionally, it was elucidated that the differences between HCC and control groups in fucosylated biomarker candidates A1AT and FETUA mainly originated from an increase in fucosylation levels on these target glycoproteins, rather than an increase in the total protein abundance of the target glycoproteins.

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. Proteolytic digestion then results in quantitatively different peptide products in accordance with the degree of glycosylation. The effect of glycan steric hindrance on tryptic digestion was first demonstrated using alpha-1-acid glycoprotein (AGP) as a model compound versus deglycosylated alpha-1-acid glycoprotein. Second, nonglycosylated tryptic peptide biomarkers, which generally show much higher sensitivity in mass spectrometric analyses than their glycosylated counterparts, were quantified in human hepatocellular carcinoma plasma using a label-free method with no need for N-linked glycoprotein enrichment. Finally, the method was validated using a multiple reaction monitoring analysis, demonstrating that the newly discovered nonglycosylated tryptic peptide targets were present at different levels in normal and hepatocellular carcinoma plasmas. The area under the receiver operating characteristic curve generated through analyses of nonglycosylated tryptic peptide from vitronectin precursor protein was 0.978, the highest observed in a group of patients with hepatocellular carcinoma. This work provides a targeted means of discovering and validating nonglycosylated tryptic peptides as biomarkers in human plasma, without the need for complex enrichment processes or expensive antibody preparations.

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 ß-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. Each lectin-captured fraction containing aberrant glycoforms of TIMP1 was digested by trypsin, resulting in the tryptic target peptide, representative of the serum glycoprotein TIMP1. The resulting target peptide was enriched using a stable isotope standard and capture by the antipeptide antibody (SISCAPA) technique and analyzed by a 15 T MALDI FTICR mass spectrometer with high mass accuracy (Δ < 0.5 ppm to the theoretical mass value of the target peptide). Since exact measurement of multiplex isotopic peaks of the target peptide could be accomplished by virtue of high mass resolution (Rs > 400,000), robust identification of the target peptide is only achievable with 15 T FTICR MS. Also, MALDI data obtained in this study showed that the L-PHA-captured glycoforms of TIMP1 were measured in the pooled CRC serum with about 5 times higher abundance than that in the noncancerous serum, and were further proved by MRM mass analysis. These results confirm that TIMP1 in human serum is a potent CRC biomarker candidate, demonstrating that ultrahigh-resolution MS can be a powerful tool toward identifying and verifying potential protein biomarker candidates.

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. Thus, it was confirmed that the multiplex lectin-channel monitoring (LCM) using multiple lectins is useful for investigating protein glycosylation diversity in a proteome sample. Based on the semi-quantitative mass profiling, target proteins showing lectin-specificity among each lectin-captured fraction were selected and analyzed by the MRM-based method in triplicate using each lectin-captured fraction (average CV 7.9%). The MRM-based analysis for each lectin-captured fraction was similar to those obtained by the profiling experiments. The abundance of each target protein measured varied dramatically, based on the lectin-specificity. The multiplex LCM approach using MRM-based analyses is useful for quantitatively monitoring target protein glycoforms selectively fractionated by multiple lectins. Thus through multiplex LCM rather than single, we could inquire minutely into protein glycosylation states.

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. Plasma samples prepared from hepatocellular carcinoma (HCC) patients without immuno-depletion of highly abundant plasma proteins were fractionated by use of fucose-specific aleuria aurantia lectin (AAL) immobilized on magnetic beads by use of a biotin-streptavidin conjugate. The lectin-captured fractions were digested by trypsin and profiled by tandem mass spectrometry. From the proteomic profiling data, target glycoproteins were selected and analyzed quantitatively by MRM-based analysis. The reproducibility of MRM-based quantification of the selected target proteins was reliable, with precision (CV; ≤14% for batch-to-batch replicates and ≤19% for replicates over three days) sufficient to distinguish differences in the abundance of AAL-captured glycoforms between individual plasma samples. This lectin-coupled, MRM-based method, measuring only lectin-captured glycoforms of a target protein rather than total target protein, is a tool for monitoring differences between individuals by measuring the abundance of aberrant glycoforms of a target protein related to a disease. This method may be further applied to rapid verification of biomarker candidates involved in aberrant protein glycosylation in human plasma.

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. The high expression ratio of galectin-3 binding protein to galectin-3 was clinically correlated to lymphatic invasion. These results suggest that galectin-3 binding protein may be a potential therapeutic target for treatment of, at least, colon cancer patients with high expression of galectin-3 binding protein.

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

Glechoma hederacea var. longituba (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. In this study, liquid chromatography-mass spectrometry technology was used for comparative analysis of constituents in hot-water extracts of GHL samples obtained from various cultivating provinces in South Korea. A set of marker panel consisting of nine polyphenolic compounds, including glucuronide conjugates in particular, was constructed and used to monitor the compositional characteristics in each GHL extract. Our findings show that some of the marker compounds, including rosmarinic acid, were persistently observed as major constituents in the analyses of the 22 GHL sample extracts, whereas, interestingly, other marker compounds such as polyphenol-glucuronic acid conjugates displayed dramatic differences in compositional ratios. This chromatographic approach using the marker compound panel can be applied to qualitatively and quantitatively evaluate compositional characteristics in the GHL extracts, and can also be useful for quality assays of the GHL herbal plant in medicinal and industrial fields.

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. Pull-down experiments using recombinant HMGB1 protein as bait, followed by mass spectrometry analysis identified 162 interacting proteins. Among them were 74 proteins known to be localized exclusively to the extra-nuclear region, and 60 proteins known to be localized to both nuclear and extranuclear regions. The functions of these binding proteins include involvement in cell-cycle progression, cell proliferation, anti-apoptosis, and angiogenesis. In addition, nine of the identified proteins are related to protein translocation and secretion. These include annexin A2, myosin IC isoform a, myosin-9, and Ras-related protein Rab10, which are involved in unconventional protein secretion. Cytoplasmic HMGB1 was primarily associated with the lysosomal cytosol fraction and was colocalized with the lysosomal marker LAMP1. Our findings suggest that cytoplasmic HMGB1 binds to a number of molecules related to cancer progression and the unconventional secretory pathway.

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. MRM quantitation data for the L-PHA-enriched samples revealed that the abundance of aberrant glycoforms of TIMP1 and PTPkappa was greatly increased (11.7- and 16.5-fold, respectively) in GnT-V-treated cells compared to the control cells, although the abundance of total TIMP1 and PTPkappa in GnT-V-treated cells was slightly different (1.1- and 0.5-fold, respectively) for unenriched samples compared to that in control cells. The dramatic variation in abundance of the aberrant glycoforms due to overexpressed GnT-V was confirmed quantitatively by comparative MRM analysis of lectin-enriched samples. This method is capable of comparatively quantitating the abundance of a protein of interest and its aberrant glycoform and will be useful for studying pathological mechanisms of cancer or verifying biomarker candidates.

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. We carried out a kinase inhibitor study and evaluated the cell migration by invasion assay. We showed that phosphorylated HMGB1 localizes in the cytoplasm of colon cancer cells and also showed the interaction of PKC and HMGB1 by immunoprecipitation analysis. Concurrent mutations at six serine residues (35, 39, 42, 46, 53, and 181) to glutamic acid induced the nuclear to cytoplasmic transport of HMGB1, which was detected in the culture medium. We also observed that the secretion of HMGB1 correlated with increased cancer cell invasiveness. Our results suggest that phosphorylated HMGB1 is transported to the cytoplasm, is subsequently secreted from the cell, and has a role in tumor progression through the activation of genes related to cell migration.

Anaerobic organic acid production of food waste in once-a-day feeding and drawing-off bioreactor.

Acidogenesis of food waste was studied in a 2-L reactor with semi-continuous mode operation (once-a-day feeding and draw-off) for maximum 65 days to examine optimal volatile acid compositions for biological nitrogen removal (BNR) and enhanced biological phosphorus removal (ENPR). Various operational parameters of hydraulic retention time (HRT), organic loading rate (ORL), pH and temperature were investigated for soluble chemical oxygen demand (SCOD), volatile fatty acid composition, nitrogen and phosphate. The yields (gTVFA/g VS) and the volumetric productivity (gTVFA/d L) increased with HRT from 0.26-0.32, 1.25-1.50 (at 4 days) to 0.36-0.39, 1.71-1.83 (at 12 days). However, the acetate fraction (%) decreased with HRT from 35.7-37.5 at 4 days to 23.5-25 at 12 days. The yields decreased with increase of organic loading from 0.34-0.37 at 5 g/L d to 0.29-0.30 at 13 g/L d and the productivity increased from 1.63-1.65 to 3.61-3.75. The yield and productivity were highest at 35 degrees C among 25, 35 and 45 degrees C. The yield and productivity at pH 5.5 and 6.0 were best and very similar to each other. The condition of 35 degrees C, pH 6.0, HRT 8 days, ORL 9 g/L d resulted in TVFA, SCOD, acetate and butyrate of 25, 39.5, 12 and 5.25 g/L, respectively.

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. Furthermore, the comparative analysis of a mutagenized rAb glycoprotein proved that a single amino acid mutation in the Fc portion of the antibody molecule caused increased variations in glycosylation patterns. These variations were also detected by the mass spectrometry method efficiently. This mapping method, focusing on precise glycopeptide identification and comparison for the identified glycoforms, can be useful in differentiating aberrant glycosylation in biosimilar rAb products.

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. A new MALDI MS data interpretation method was developed, consisting of a peak-resolving algorithm for overlapping peaks in MALDI MS spectra, a retrosynthetic analysis for the generation of reduced unit mass peaks, and a Gaussian fit-based selection of the most prominent polymer series among the reconstructed unit mass peaks. This method of data interpretation avoids errors originating from side reactions due to the presence of trace water in the reaction mixture or MALDI analysis. Quantitative changes in the relative compositions of the resulting polymer products were monitored as a function of reaction time. These results demonstrate that the mass data interpretation method described herein can be a powerful tool for estimating quantitative changes in the compositions of polymer products arising during a polymerization reaction.

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. By integrating multiple transition signals corresponding to fragment ions in the full scan MS/MS spectrum of a precursor ion of the target peptide, a pseudo-MRM MS analysis of the target peptide showed an increased signal-to-noise (S/N) ratio and sensitivity, as well as an improved reproducibility. The pseudo-MRM method was then used for the quantitative analysis of the tryptic peptides of two low-abundance serological proteins, tissue inhibitor of metalloproteinase 1 (TIMP1) and tissue-type protein tyrosine phosphatase kappa (PTPκ), which were prepared with peptide affinity-based enrichment from human serum. Finally, this method was used to detect femtomolar amounts of target peptides derived from TIMP1 and PTPκ, with good coefficients of variation (CV 2.7% and 9.8%, respectively), using a few microliters of human serum from colorectal cancer patients. The results suggest that pseudo-MRM using hybrid Q-TOF MS, combined with peptide affinity-based enrichment, could become a promising alternative for the quantitative analysis of low-abundance target proteins of interest in complex serum samples that avoids protein depletion.