The analysis of alphaâ 1â antitrypsin glycosylation with direct LCâ MS/MS

A liquid chromatographyâ tandem mass spectrometry (LCâ MS/MS)â based methodology has been developed to differentiate coreâ and antennaryâ fucosylated glycosylation of glycopeptides. Both the glycosylation sites (heterogeneity) and multiple possible glycan occupancy at each site (microheterogeneity) can be resolved via intact glycopeptide analysis. The serum glycoprotein alphaâ 1â antitrypsin (A1AT) which contains both coreâ and antennaryâ fucosylated glycosites was used in this study. Sialidase was used to remove the sialic acids in order to simplify the glycosylation microheterogeneity and to enhance the MS signal of glycopeptides with similar glycan structures. β1â 3,4 galactosidase was used to differentiate coreâ and antennaryâ fucosylation. Inâ source dissociation was found to severely affect the identification and quantification of glycopeptides with low abundance glycan modification. The settings of the mass spectrometer were therefore optimized to minimize the inâ source dissociation. A threeâ step mass spectrometry fragmentation strategy was used for glycopeptide identification, facilitated by pGlyco software annotation and manual checking. The collision energy used for initial glycopeptide fragmentation was found to be crucial for improved detection of oxonium ions and better selection of Y1 ion (peptide+GlcNAc). Structural assignments revealed that all three glycosylation sites of A1AT glycopeptides contain complex Nâ glycan structures: site Asn70 contains biantennary glycans without fucosylation; site Asn107 contains biâ , triâ and tetraâ antennary glycans with both coreâ and antennaryâ fucosylation; site Asn271 contains biâ and triâ antennary glycans with both coreâ and antennaryâ fucosylation. The relative intensity of coreâ and antennaryâ fucosylation on Asn107 was similar to that of the A1AT protein indicating that the glycosylation level of Asn107 is much larger than the other two sites.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/146302/1/elps6432_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/146302/2/elps6432.pd

Discrimination of delays of reinforcement in aversive conditioning.

<p>(a) The protein level changes of CA125 and LRG1 were confirmed in the larger sample set 2. (b) Marker distributions for the EOC patients with different histological subtypes.</p

An in vitro vesicle formation assay reveals cargo clients and factors that mediate vesicular trafficking

The fidelity of protein transport in the secretory pathway relies on the accurate sorting of proteins to their correct destinations. To deepen our understanding of the underlying molecular mechanisms, it is important to develop a robust approach to systematically reveal cargo proteins that depend on specific sorting machinery to be enriched into transport vesicles. Here, we used an in vitro assay that reconstitutes packaging of human cargo proteins into vesicles to quantify cargo capture. Quantitative mass spectrometry (MS) analyses of the isolated vesicles revealed cytosolic proteins that are associated with vesicle membranes in a GTP-dependent manner. We found that two of them, FAM84B (also known as LRAT domain containing 2 or LRATD2) and PRRC1, contain proline-rich domains and regulate anterograde trafficking. Further analyses revealed that PRRC1 is recruited to endoplasmic reticulum (ER) exit sites, interacts with the inner COPII coat, and its absence increases membrane association of COPII. In addition, we uncovered cargo proteins that depend on GTP hydrolysis to be captured into vesicles. Comparing control cells with cells depleted of the cargo receptors, SURF4 or ERGIC53, we revealed specific clients of each of these two export adaptors. Our results indicate that the vesicle formation assay in combination with quantitative MS analysis is a robust and powerful tool to uncover novel factors that mediate vesicular trafficking and to uncover cargo clients of specific cellular factors.</p

Extracellular Proteome Analysis of Leptospira interrogans serovar Lai

Leptospirosis is one of the most important zoonoses. Leptospira interrogans serovar Lai is a pathogenic spirochete that is responsible for leptospirosis. Extracellular proteins play an important role in the pathogenicity of this bacterium. In this study, L. interrogans serovar Lai was grown in protein-free medium; the supernatant was collected and subsequently analyzed as the extracellular proteome. A total of 66 proteins with more than two unique peptides were detected by MS/MS, and 33 of these were predicted to be extracellular proteins by a combination of bioinformatics analyses, including Psortb, cello, SoSuiGramN and SignalP. Comparisons of the transcriptional levels of these 33 genes between in vivo and in vitro conditions revealed that 15 genes were upregulated and two genes were downregulated in vivo compared to in vitro. A BLAST search for the components of secretion system at the genomic and proteomic levels revealed the presence of the complete type I secretion system and type II secretion system in this strain. Moreover, this strain also exhibits complete Sec translocase and Tat translocase systems. The extracellular proteome analysis of L. interrogans will supplement the previously generated whole proteome data and provide more information for studying the functions of specific proteins in the infection process and for selecting candidate molecules for vaccines or diagnostic tools for leptospirosis.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/140174/1/omi.2013.0043.pd

Label‐free relative quantification of alpha‐2‐macroglobulin site‐specific core‐fucosylation in pancreatic cancer by LC‐MS/MS

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/108096/1/elps4971.pd

Analysis of Glycan Variation on Glycoproteins from Serum by the Reverse Lectin-Based ELISA Assay

Altered glycosylation in glycoproteins is associated with carcinogenesis, and certain glycan structures and glycoproteins are well-known markers for tumor progression. To identify potential diagnostic candidate markers, we have developed a novel method for analysis of glycosylation changes of glycoproteins from crude serum samples using lectin-based glycoprotein capture followed by detection with biotin/HRP-conjugated antibodies. The amount of lectin coated on the microplate well was optimized to achieve low background and improved S/N compared with current lectin ELISA methods. In the presence of competing sugars of lectin AAL or with sialic acid removed from the glycoproteins, we confirmed that this method specifically detects glycosylation changes of proteins rather than protein abundance variation. Using our reverse lectin-based ELISA assay, increased fucosylated haptoglobin was observed in sera of patients with ovarian cancer, while the protein level of haptoglobin remained the same between cancers and noncases. The combination of fucosylated haptoglobin and CA125 (AUC = 0.88) showed improved performance for distinguishing stage-III ovarian cancer from noncases compared with CA125 alone (AUC = 0.86). In differentiating early-stage ovarian cancer from noncases, fucosylated haptoglobin showed comparable performance to CA125. The combination of CA125 and fucosylated haptoglobin resulted in an AUC of 0.855, which outperforms CA125 to distinguish early-stage cancer from noncases. Our study provides an alternative method to quantify glycosylation changes of proteins from serum samples, which will be essential for biomarker discovery and validation studies