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Detection and Characterization of Post-Translational Modifications in AL Light Chain Proteins by Mass Spectrometry
Published in Gilles Grateau, Robert A. Kyle, Martha Skinner, Amyloid and Amyloidosis, 2004
R. Théberge, A. Lim, Y. Jiang, LH Connors, J. Eberhard, M. Skinner, CE Costello
Reduction was performed with a 10-fold molar excess of DTT over expected disulfides (100 mM NH4HCO3 pH 8, 30 min, 37 °C). Asp-N (enzyme:substrate, 1:300) digestion was carried out in 100 mM NH4HCO3 (pH 8, 37 °C, 5 hr). Lys-C (1:100) digestion was performed in 100 mM NH4HCO3 (pH 8, 37 °C, 7 hr). ESI mass spectra of the intact proteins before and after reduction were obtained in the positive-ion mode using a Micromass Quattro II triple quadrupole MS. All enzymatic digests were mixed with 2,5-dihydroxybenzoic acid and analyzed with a Finnigan MAT Vision 2000 MALDI time-of-flight (TOF) MS, using delayed extraction in the linear mode. PNGase F was used to release N-linked glycans from the glycosylated light chain. The released glycans were permethylated using standard protocol and analyzed using the Reflex IV MALDI-TOF MS. Peptides of interest were sequenced with an Applied Biosystems/MDS-SCIEX QSTARTM Pulsar quadrupole/orthogonal acceleration TOF MS.
Biochemical Analysis of the Polycystin-1 Complexity Generated by Proteolytic Cleavage at the G Protein-Coupled Receptor Proteolysis Site
Published in Jinghua Hu, Yong Yu, Polycystic Kidney Disease, 2019
Rebecca Walker, Hangxue Xu, Qiong Huang, Feng Qian
One important consideration is the heavy N-glycosylation of PC1cFL and PC1deN in tissues, which significantly increases their molecular weight and gives rise to their doublet bands on Western blot. In the case of PC1cFL, this causes the upper band of the PC1NTF doublet to migrate to a position that partially overlaps with that of the PC1U, obscuring the distinction between the two PC1 forms even under optimized polyacrylamide gel electrophoresis conditions. This issue can be resolved by N-glycosylation analysis using the N-deglycosylases PNGase-F (peptide N-glycosidase F) and Endo-H (endoglycosidase H)9,12,69,70 (see Protocol III). PNGase-F removes all types of N-linked glycans (high mannose, hybrid, and complex N-glycans) from glycoproteins, while Endo-H removes only high mannose and some hybrid types of N-linked carbohydrates. The use of the two N-deglycosylases not only helps to distinguish between PC1U from of PC1cFL and PC1deN in tissues and cells, but also serves to monitor their trafficking along the secretory pathway.69,70 The general rationale is that N-glycans of glycoproteins in the ER are all high mannose and are susceptible to removal by cleavage using PNGase-F and Endo-H, whereas complex N-glycans acquired in the medial/trans-Golgi compartment are resistant to removal by Endo-H but remain sensitive to PNGase-F. Sensitivity to Endo-H is therefore indicative of proteins that are still in the ER, whereas proteins that acquire Endo-H resistance have egressed the ER and passed through the Golgi compartment.
Integration of transcriptomic and proteomic approaches for snake venom profiling
Published in Expert Review of Proteomics, 2021
Cassandra M. Modahl, Anthony J. Saviola, Stephen P. Mackessy
An area of venom proteomics that has been neglected is the use of MS approaches to evaluate venom protein glycosylations. N-linked glycan structures have been found to be present in many venoms, especially those of Viperidae [62], but venoms from most species have not been studied. Standard bottom-up workflows that are currently the more common venomics approaches cannot be used to examine glycosylations because collisional activation does not usually cleave glycosylated peptides, making them harder to detect. In these instances, the initial removal of N-linked glycans by PNGase F could aid in both identification of these glycosylated sites and better protein coverage. The use of multienzyme digestions also offers a means to increase the number of different peptide fragments that can be identified, and this method would increase coverage and accuracy in determining venom proteoforms present [63]. Integration of transcriptome databases will aid both of these techniques, as glycosylation sites can be predicted from translated transcriptomes and provide proteoform reference sequences. These future directions in venomics pipelines will provide a more comprehensive picture of snake venom proteins and post-translational modifications of these potent biomolecules.
Altered biodistribution of deglycosylated extracellular vesicles through enhanced cellular uptake
Published in Journal of Extracellular Vesicles, 2020
Nao Nishida-Aoki, Naoomi Tominaga, Nobuyoshi Kosaka, Takahiro Ochiya
The impact of glycosylation subtypes on EV uptake was further analysed using EVs from BMD2a cells. PKH67-labelled EVs from BMD2a were treated by either N- or O-glycosylation degrading enzymes. Peptide-N-Glycosidase F (PNGase F) removes almost all N-linked glycans from glycoproteins. For O-deglycosylation, EVs were treated with a mixture of Neuraminidase and O-glycosidase because Neuraminidase removes sialic acids, which is a structural obstacle for O-glycosidase activities. Glycosylation patterns of EVs after treated with deglycosylation enzymes were analysed by lectin blots to confirm that glycosylations were removed (Supplementary Fig. 4A). Decreased molecular weight or decreased staining intensity was observed after deglycosylation, supporting that the activity of the deglycosylation enzymes. The particle size distribution and the number of BMD2a EVs after deglycosylation treatments remained mostly the same; therefore, EVs population compromised with vesicle structures after enzymatic treatment is ignorable (Supplementary Fig. 4B). The uptake of N- or O-glycosylation-deprived BMD2a EVs to HUVECs was tested in the same procedure as explained. N-, O-, or N-and O-glycosylation removal further increased BMD2a EVs uptake to HUVECs to similar levels (Figure 2b, Supplementary Fig. 3C). This result indicated that both N- and O- glycosylation on the surface of EVs have an inhibitory effect on EVs uptake in vitro.
A novel human monoclonal antibody specific to the A33 glycoprotein recognizes colorectal cancer and inhibits metastasis
Published in mAbs, 2020
Patrizia Murer, Louis Plüss, Dario Neri
The cDNA encoding for the A33 construct was obtained by total RNA extraction from the HT-29 colorectal carcinoma cell line (High Pure RNA Isolation Kit, Roche), reverse transcription (Transcriptor reverse transcriptase, Roche) and amplification of A33 cDNA by Taq DNA Polymerase (Sigma-Aldrich). The extracellular domains of the antigen were PCR amplified, extended with a nucleic acid sequence encoding for six histidine residues (His-tag) and cloned into the mammalian expression vector pcDNA3.1(+) (Invitrogen). The soluble antigen was produced by transient gene expression in CHO cells as described previously42 and purified from the cell culture medium by Ni-NTA resin (Roche). Quality control of A33-His was performed by SDS-PAGE and size exclusion chromatography (Superdex75 10/300GL, GE Healthcare). The protein was digested with PNGase F (New England BioLabs) under denaturing conditions. A33-His was biotinylated with Sulfo-NHS-LC-Biotin (Pierce). Biotinylation of the antigen was tested through mass spectrometry (MS) analysis and band shift assay [Supplementary Figure 5].