Sz-Wei Wu

Frequent epidermal growth factor receptor gene mutations in malignant pleural effusion of lung adenocarcinoma

Malignant pleural effusions (MPEs) are often observed in lung cancer, especially adenocarcinoma. Epidermal growth factor receptor (EGFR) gene mutations are usually detected in lung adenocarcinoma. The purpose of the present study was to investigate the EGFR mutation rate in MPEs of lung adenocarcinoma. Between June 2005 and December 2006, 136 MPEs from lung adenocarcinoma were collected for EGFR mutation detection. In addition, between April 2001 and November 2004, 91 surgically resected specimens of lung adenocarcinoma from patients without MPEs were assessed for EGFR mutation. The EGFR mutation rate was significantly higher in the patients with MPEs than in the patients without (68.4% versus 50.5%). The EGFR mutation rate in patients with MPEs was not associated with sex, smoking history, age or cancer stage. By multivariate analysis, an age of <65 yrs, never smoking, Eastern Cooperative Oncology Group performance status 0–1, and EGFR mutation were significantly associated with a longer overall survival for lung adenocarcinoma patients with MPEs. The patients with malignant pleural effusions related to lung adenocarcinoma had a higher epidermal growth factor receptor gene mutation rate than the patients from whom surgically resected specimens were taken. Epidermal growth factor receptor tyrosine kinase inhibitors may be the treatment of choice for lung adenocarcinoma with malignant pleural effusions in east Asia.

Distinctive characteristics of MALDI-Q/TOF and TOF/TOF tandem mass spectrometry for sequencing of permethylated complex type N-glycans.

Concerted MALDI-MS profiling and CID MS/MS sequencing of permethylated glycans is one of the most effective approaches for high throughput glycomics applications. In essence, the identification of larger complex type N-glycans necessitates an unambiguous definition of any modification on the trimannosyl core and the complement of non-reducing terminal sequences which constitute the respective antennary structures. Permethylation not only affords analyses of both neutral and sialylated glycans at comparable ease and sensitivity but also yields more sequence-informative fragmentation pattern. Facile glycosidic cleavages directed mostly at N-acetylglucosamine under low energy CID, as implemented on a quadrupole/time-of-flight (Q/TOF) instrument, often afford multiple losses of the attached antenna resulting in characteristic ions related to the number of antennary branches on the trimannosyl core. Non-reducing terminal epitopes can be easily deduced but information on the linkage specific substituent on the terminal units is often missing. The high energy CID MS/MS afforded by TOF/TOF instrument can fill in the gap by giving an array of additional cross-ring and satellite ions. Glycosidic cleavages occurring specifically in concert with loss of 2-linked or 3-linked substituents provide an effective way to identify the branch-specific antennary extension. These characteristics are shown here to be effective in deriving the sequences of additionally galactosylated, sialylated and fucosylated terminal N-acetyllactosamine units and their antennary location. Together, a highly reproducible fragmentation pattern can be formulated to simplify spectral assignment. This work also provides first real examples of sequencing multiply sialylated complex type N-glycans by high energy CID on a TOF/TOF instrument.

Enabling techniques and strategic workflow for sulfoglycomics based on mass spectrometry mapping and sequencing of permethylated sulfated glycans

Sulfate modifications on terminal epitopes of N- and O-glycans have increasingly been implicated as critical determinants mediating a diverse range of biological recognition functions. To address these low abundance but important sulfated glycans, and the sulfoglycome in general, further development of enrichment strategies and enabling mass spectrometry (MS)-based mapping techniques are needed. In this report, we demonstrate that the sulfated glycans, with and without additional sialylation, can be successfully permethylated by the sodium hydroxide slurry method and be distinguished from phosphorylated glycans by virtue of this derivatization. In conjunction with simple microscale postderivatization fractionation steps, permethyl derivatives fully retaining the negatively charged sulfate moiety and separated from the nonsulfated ones, can be efficiently detected and sequenced de novo by advanced MS/MS in the positive-ion mode. In particular, we show that the highly sequence and linkage informative high energy collision induced dissociation (CID) MS/MS afforded by MALDI-TOF/TOF can be extended to sulfoglycomic applications. The sulfated parent ion selected for CID MS/MS was found to mostly retain the sulfate moiety and therefore allow efficient fragmentation via the usual array of glycosidic, cross ring, and concerted double cleavages. Collectively, the optimized strategy enables a high sensitivity detection and critical mapping of the sulfoglycome such as the one derived from lymph node tissues or cell lines in both negative and positive-ion modes. Novel sulfated epitopes were identified from a crude mouse lymph node preparation, which fully attested to the practical utility of the methodology developed.

Novel LC-MS2 Product Dependent Parallel Data Acquisition Function and Data Analysis Workflow for Sequencing and Identification of Intact Glycopeptides

Data dependent acquisition (DDA) of higher collision energy dissociation (HCD)-MS(2) followed by electron transfer dissociation (ETD)-MS(2) upon detection of glycan-specific oxonium is one of the better approaches in current LC-MS(2) analysis of intact glycopeptides. Although impressive numbers of glycopeptide identification by a direct database search have been reported, false positives remained high and difficult to determine. Even in cases when the peptide backbones were correctly identified, the exact glycan moieties were often erroneously assigned. Any attempt to fit the best glycosyl composition match by mass only is problematic particularly when the correct monoisotopic precursor cannot be determined unambiguously. Taking advantage of a new trihybrid Orbitrap configuration, we experimented with adding in a parallel ion trap collision induced dissociation (CID)-MS(2) data acquisition to the original HCD-product dependent (pd)-ETD function. We demonstrated the feasibility and advantage of identifying the peptide core ion directly from edited HCD-MS(2) data as an easy way to reduce false positives without compromising much sensitivity in intact glycopeptide positive spectrum matches. Importantly, the additional CID-MS(2) data allows one to validate the glycan assignment and provides insight into possible glycan modifications. Moreover, it is a viable alternative to deduce the glycopeptide backbone particularly in cases when the peptide backbone cannot be identified by ETD/HCD. The novel HCD-pd-CID/ETD workflow combines the best possible decision tree dependent MS(2) data acquisition modes currently available for glycoproteomics within a rapid Top Speed DDA duty cycle. Additional informatics can conceivably be developed to mine and integrate the rich information contained within for simultaneous N- and O-glycopeptide analysis.

Glycoproteomics analysis to identify a glycoform on haptoglobin associated with lung cancer

Glycosylation is a common protein modification that is of interest in current cancer research because altered carbohydrate moieties are often found during cancer progress. A search for biomarkers in human lung cancer serum samples using glycoproteomic approaches identified fucosylated haptoglobin (Hp) significantly increased in serum of each subtype of lung cancer compared to normal donors. In addition, MS provided evidence of an increase of Hp fucosylation; the glycan structure was determined to be an α 2,6‐linked tri‐sialylated triantennary glycan containing α1,3‐linked fucose attached to the four‐linked position of the three‐arm mannose of N‐linked core pentasaccharide. These preliminary findings suggest that the specific glycoform of Hp may be useful as a marker to monitor lung cancer progression.

AGO61-dependent GlcNAc modification primes the formation of functional glycans on α-dystroglycan.

Dystroglycanopathy is a major class of congenital muscular dystrophy that is caused by a deficiency of functional glycans on α-dystroglycan (α-DG) with laminin-binding activity. A product of a recently identified causative gene for dystroglycanopathy, AGO61, acted in vitro as a protein O-mannose β-1, 4-N-acetylglucosaminyltransferase, although it was not functionally characterized. Here we show the phenotypes of AGO61-knockout mice and demonstrate that AGO61 is indispensable for the formation of laminin-binding glycans of α-DG. AGO61-knockout mouse brain exhibited abnormal basal lamina formation and a neuronal migration defect due to a lack of laminin-binding glycans. Furthermore, our results indicate that functional α-DG glycosylation was primed by AGO61-dependent GlcNAc modifications of specific threonine-linked mannosyl moieties of α-DG. These findings provide a key missing link for understanding how the physiologically critical glycan motif is displayed on α-DG and provides new insights on the pathological mechanisms of dystroglycanopathy.

Terminal disialylated multiantennary complex-type N-glycans carried on acutobin define the glycosylation characteristics of the Deinagkistrodon acutus venom

Glycosylation analysis of nonmammalian sources often springs surprises and conjures up intriguing views of evolutionary adaptation. Many of the constituents of snake venoms are known to be glycosylated and yet very few were fully characterized and accorded specific functions. In the process of glycomic screening through the venoms from Asian pit vipers, a partially O-acetylated NeuAcα2-8NeuAcα2-3Galβ1-4GlcNAcβ1-terminal epitope was found to be the predominant glycosylation characteristic of the snake venom produced by the monotypic Deinagkistrodon acutus, with acutobin, a highly specific fibrinogenase, being identified as a primary protein carrier. Full structural definition and glycosylation site mapping were completed through advanced mass spectrometry analyses at both the glycan and glycopeptide levels in conjunction with chemical and enzymatic cleavages. Although similar occurrence of such terminal disialyl cap on the N-glycans of several mammalian glycoproteins has been implicated, most of these correspond to only minor constituents of the full glycomic heterogeneity and remain poorly characterized. In contrast, each antennae of the hybrid- and complex-type N-glycans derived from acutobin was found to be rather homogeneously disialylated. With up to eight sialic acids evenly distributed on nonextended tetraantennary core structure, these unusual N-glycans are among those of highest sialic acid density ever identified without actually carrying polysialic acid chains. It remains to be tested whether they may serve as multivalent disialyl ligands for several of the human Siglecs and thus meddle with the natural immuno-recognition systems of snakebite victims, apart from affecting the general efficacy of acutobin as anticoagulant in biomedical applications.

A single arabinan chain is attached to the phosphatidylinositol mannosyl core of the major immunomodulatory mycobacterial cell envelope glycoconjugate, lipoarabinomannan.

Background: Important details of the structures of lipomannan and lipoarabinomannan remain unknown. Results: New details on the branching structure of LM and the number of arabinan chains in LAM are here provided. Conclusion: Mature LAM carries a single arabinan chain attached near the middle of the mannan core. Significance: Results allow for a working model of the biosynthetic pathway of LM and LAM. Lipoarabinomannan (LAM) is composed of a phosphatidylinositol anchor followed by a mannan followed by an arabinan that may be capped with various motifs including oligosaccharides of mannose. A related polymer, lipomannan (LM), is composed of only the phosphatidylinositol and mannan core. Both the structure and the biosynthesis of LAM have been studied extensively. However, fundamental questions about the branching structure of LM and the number of arabinan chains on the mannan backbone in LAM remain. LM and LAM molecules produced by three different glycosyltransferase mutants of Mycobacterium smegmatis were used here to investigate these questions. Using an MSMEG_4241 mutant that lacks the α-(1,6)-mannosyltransferase used late in LM elongation, we showed that the reducing end region of the mannan that is attached to inositol has 5–7 unbranched α-6-linked-mannosyl residues followed by two or three α-6-linked mannosyl residues branched with single α-mannopyranose residues at O-2. After these branched mannosyl residues, the α-6-linked mannan chain is terminated with an α-mannopyranose at O-2 rather than O-6 of the penultimate residue. Analysis of the number of arabinans attached to the mannan core of LM in two other mutants (ΔembC and ΔMSMEG_4247) demonstrated exactly one arabinosyl substitution of the mannan core suggestive of the arabinosylation of a linear LM precursor with ∼10–12 mannosyl residues followed by additional mannosylation of the core and arabinosylation of a single arabinosyl “primer.” Thus, these studies suggest that only a single arabinan chain attached near the middle of the mannan core is present in mature LAM and allow for an updated working model of the biosynthetic pathway of LAM and LM.

An adaptive workflow coupled with Random Forest algorithm to identify intact N-glycopeptides detected from mass spectrometry

MOTIVATION Despite many attempts for algorithm development in recent years, automated identification of intact glycopeptides from LC-MS(2) spectral data is still a challenge in both sensitivity and precision. RESULTS We implemented a supervised machine learning algorithm, Random Forest, in an automated workflow to identify N-glycopeptides using spectral features derived from ion trap-based LC-MS(2) data. The workflow streamlined high-confident N-glycopeptide spectral data and enabled adaptive model optimization with respect to different sampling strategies, training sample size and feature set. A critical evaluation of the features important for glycopeptide identification further facilitated effective feature selection for model improvement. Using split sample testing method from 577 high-confident N-glycopeptide spectral data, we demonstrated that an optimal true-positive rate, precision and false-positive rate of 73, 88 and 10%, respectively, can be attained for overall N-glycopeptide identification Availability and implementation: The workflow developed in this work and the application suite, Sweet-Heart, that the workflow supports for N-glycopeptide identification are available for download at http://sweet-heart.glycoproteomics.proteome.bc.sinica.edu.tw/.

Selective Extraction and Effective Separation of Galactosylsphingosine (Psychosine) and Glucosylsphingosine from Other Glycosphingolipids in Pathological Tissue Samples

To facilitate the study of the chemical pathology of galactosylsphingosine (psychosine, GalSph) in Krabbe disease and glucosylsphingosine (GlcSph) in Gaucher disease, we have devised a facile method for the effective separation of these two glycosylsphingosines from other glycosphingolipids (GSLs) in Krabbe brain and Gaucher spleen samples. The procedure involves the use of acetone to selectively extract GalSph and GlcSph, respectively, from Krabbe brain and Gaucher spleen samples. Since acetone does not extract other GSLs except modest amounts of galactosylceramide, sulfatide, and glucosylceramide, the positively charged GalSph or GlcSph in the acetone extract can be readily separated from other GSLs by batchwise cation-exchange chromatography using a Waters Accell Plus CM Cartridge. GalSph or GlcSph enriched by this simple procedure can be readily analyzed by thin-layer chromatography or high-performance liquid chromatography.