Niagara, County of and Niagara County White Collar Employee Unit, CSEA Local 1000, AFSCME, AFL-CIO, Local 832 (2012) (MOA)

Liquid chromatography–tandem mass spectrometry (LC–MS/MS) and multiple reaction monitoring mass spectrometry (MRM-MS) proteomics analyses were performed on eccrine sweat of healthy controls, and the results were compared with those from individuals diagnosed with schizophrenia (SZ). This is the first large scale study of the sweat proteome. First, we performed LC–MS/MS on pooled SZ samples and pooled control samples for global proteomics analysis. Results revealed a high abundance of diverse proteins and peptides in eccrine sweat. Most of the proteins identified from sweat samples were found to be different than the most abundant proteins from serum, which indicates that eccrine sweat is not simply a plasma transudate and may thereby be a source of unique disease-associated biomolecules. A second independent set of patient and control sweat samples were analyzed by LC–MS/MS and spectral counting to determine qualitative protein differential abundances between the control and disease groups. Differential abundances of selected proteins, initially determined by spectral counting, were verified by MRM-MS analyses. Seventeen proteins showed a differential abundance of approximately 2-fold or greater between the SZ pooled sample and the control pooled sample. This study demonstrates the utility of LC–MS/MS and MRM-MS as a viable strategy for the discovery and verification of potential sweat protein disease biomarkers

Multiple Reaction Monitoring Mass Spectrometry for the Discovery and Quantification of O‑GlcNAc-Modified Proteins

O-linked <i>N</i>-acetylglucosamine (O-GlcNAc) is a post-translational modification regulating proteins involved in a variety of cellular processes and diseases. Unfortunately, O-GlcNAc remains challenging to detect and quantify by shotgun mass spectrometry (MS) where it is time-consuming and tedious. Here, we investigate the potential of Multiple Reaction Monitoring Mass Spectrometry (MRM-MS), a targeted MS method, to detect and quantify native O-GlcNAc modified peptides without extensive labeling and enrichment. We report the ability of MRM-MS to detect a standard O-GlcNAcylated peptide and show that the method is robust to quantify the amount of O-GlcNAcylated peptide with a method detection limit of 3 fmol. In addition, when diluted by 100-fold in a trypsin-digested whole cell lysate, the O-GlcNAcylated peptide remains detectable. Next, we apply this strategy to study glycogen synthase kinase-3 beta (GSK-3β), a kinase able to compete with O-GlcNAc transferase and modify identical site on proteins. We demonstrate that GSK-3β is itself modified by O-GlcNAc in human embryonic stem cells (hESC). Indeed, by only using gel electrophoresis to grossly enrich GSK-3β from whole cell lysate, we discover by MRM-MS a novel O-GlcNAcylated GSK-3β peptide, bearing 3 potential O-GlcNAcylation sites. We confirm our finding by quantifying the increase of O-GlcNAcylation, following hESC treatment with an O-GlcNAc hydrolase inhibitor. This novel O-GlcNAcylation could potentially be involved in an autoinhibition mechanism. To the best of our knowledge, this is the first report utilizing MRM-MS to detect native O-GlcNAc modified peptides. This could potentially facilitate rapid discovery and quantification of new O-GlcNAcylated peptides/proteins

Production of Functional Soluble Dectin-1 Glycoprotein Using an IRES-Linked Destabilized-Dihydrofolate Reductase Expression Vector

<div><p>Dectin-1 (CLEC7A) is a C-type lectin receptor that binds to β-glucans found in fungal cell walls to act as a major pattern recognition receptor (PRR). Since β-glucans epitope is not present in human cells, we are of the opinion that Dectin-1 can have therapeutic functions against fungal infections. We thus set out to produce a soluble extracellular domain of murine Dectin-1 (called sDectin-1) in sufficient titers to facilitate such studies in mouse models. Since sDectin-1 has previously been shown to be glycosylated, we chose to produce this protein using Chinese Hamster Ovary (CHO) cells, a mammalian host cell line suitable for the high-titer production of recombinant glycoproteins. To ensure a high titer production of sDectin-1 and minimize the effects of gene fragmentation, we constructed a mammalian expression vector with a PEST-destabilized dhfr amplifiable marker downstream of an attenuated IRES element, which was in turn downstream of the sDectin-1 gene and a CMV IE promoter. Stably transfected and MTX-amplified cell pools were generated using this vector, and maximum sDectin-1 titers of 246 mg/l and 598 mg/l were obtained in shake flask batch culture and bioreactor fed-batch culture respectively. The purified recombinant sDectin-1 was shown to be glycosylated. Protein functionality was also demonstrated by its ability to bind to zymosan particles and to the cell wall of <em>Saccharomyces cerevisiae</em>. We describe for the first time the use of an attenuated IRES-linked PEST-destabilized dhfr amplifiable marker for the production of recombinant proteins with stably amplified cell pools. With our process, we reached the highest reported titer for producing recombinant proteins smaller than 50 kDa in cell pools. sDectin-1 protein produced is glycosylated and functional. This vector design can thus be used efficiently for the high-titer production of functional recombinant proteins.</p> </div

Binding of cHis-sDectin-1 to <i>Saccharomyces cerevisiae</i> cells.

<p>Purified cHis-sDectin-1 was diluted to a concentration of 25 µg/ml in blocking buffer with 500 µg/ml, 100 µg/ml or no laminarin. This was added to <i>Saccharomyces cerevisiae</i> yeast cells from overnight culture in blocking buffer (PBS with 3% FBS). The cells were then probed with an mDectin-1 goat polyclonal antibody (1∶200; AF1756; R&D Systems) and AlexaFluor546-conjugated anti-goat antibody (1∶100; Catalog Number A-11056; Molecular Probes). After which, the cells were washed with PBS and fixed using 4% paraformaldehyde. The cells were then resuspended in PBS and visualized by phase contrast and fluorescent microscopy at (A) 200× magnification and (B) 600× magnification. Images are cropped or scaled to fit the illustration. cHis-sDectin-1 stained yeast cells was also analyzed by flow cytometry (C).</p

Design of sDectin-1 expression vector.

<p>(A) Vector map of pDec1-nHis and pDec1-cHis, and illustration of its design to enhance sDectin-1 production. (B) Amino acid sequence of mDectin-1 from bone-marrow derived macrophage cells of C57BL/6 mice. The peptide fragment expressed in sDectin-1, the transmembrane region, the C-type lectin domain and the two potential N-linked glycosylation sites are indicated on the sequence based on alignment to protein sequence from Uniprot Accession Q6QLQ4.</p

MTX amplification and characterization of sDectin-1 cell pools.

<p>(A) Western blotting of supernatant samples from cultures adapted to different MTX concentrations using an mDectin-1 goat polyclonal antibody (1∶1000; AF1756; R&D Systems) with a HRP conjugated anti-goat antibody (1∶2000; Catalog number V8051; Promega). cHis pool and nHis pool are cell pools producing sDectin-1 with histidine tagged at the C- and N-terminals respectively. (B) Shake flask batch culture cell growth and sDectin-1 production profiles of sDectin-1 producing cell pools. Values shown represent mean values obtained from three replicate flasks. Error bars indicate the standard deviation of the experiment.</p

Structural analysis of cHis-sDectin-1.

<p>(A) Western blot analysis of untreated and PNGase F-treated cHis-sDectin-1 produced from CHO cell pool, compared to that produced by <i>E. coli</i>, using a horseradish peroxidase (HRP)-conjugated His-tag antibody (1∶ 2000; Catalog number 71840; Merck KGaA) (B) MALDI-TOF mass spectrometry analysis of the permethylated N-glycans released from the purified sDectin-1 produced from cHis CHO cell pool. Solid square, N-acetylglucosamine; solid circle, mannose; open circle, galactose; solid triangle, fucose; solid diamond, N-acetylneuraminic acid; open diamond, N-glycolylneuraminic acid.</p

Purification of cHis-sDectin-1.

<p>cHis-sDectin-1 was purified using an IMAC nickel column and buffer exchanged using a 10 kDa molecular weight cut-off ultrafiltration spin filter. The unpurified supernatant, flow-through and eluate from the IMAC column, as well as the filtrate from the spin filter were separated by SDS-PAGE and stained using (A) Coomassie and (B) silver staining.</p

Bioreactor fed-batch production of cHis-sDectin-1 using the cHis cell pool in 500 nM MTX.

<p>(A) Cell growth and cHis-sDectin-1 production profiles in 2 L stirred tank bioreactor. (B) Metabolite profiles of the bioreactor culture.</p

Binding of zymosan to sDectin-1.

<p>20 or 200 ng cHis-sDectin-1 were coated on Maxisorp plates followed by FITC-zymosan. The plate was washed with PBS and imaged using a fluorescent microscope. Representative images from duplicate experiment are shown here.</p