ABSTRACT
Hydrogen/deuterium exchange monitored by mass spectrometry (HDX-MS) has become an important method to study the structural dynamics of proteins. However, glycoproteins represent a challenge to the traditional HDX-MS workflow for determining the deuterium uptake of the protein segments that contain the glycan. We have recently demonstrated the utility of the glycosidase PNGase A to enable HDX-MS analysis of N-glycosylated protein regions. Here, we have investigated the use of the acidic glycosidase PNGase H+, which has a pH optimum at 2.6, to efficiently deglycosylate N-linked glycosylated peptides during HDX-MS analysis of glycoproteins. Our results show that PNGase H+ retains high deglycosylation activity at HDX quench conditions. When used in an HDX-MS workflow, PNGase H+ allowed the extraction of HDX data from all five glycosylated regions of the serpin α1-antichymotrypsin. We demonstrate that PNGase A and PNGase H+ are capable of similar deglycosylation performance during HDX-MS analysis of α1-antichymotrypsin and the IgG1 antibody trastuzumab (TZ). However, PNGase H+ provides broader specificity and greater tolerance to the disulfide-bond reducing agent TCEP, while PNGase A offers advantages in terms of commercial availability and purity. Overall, our findings demonstrate the unique features of PNGase H+ for improving conformational analysis of glycoproteins by HDX-MS, in particular, challenging glycoproteins containing both glycosylations and disulfide bonds.
Subject(s)
Amidohydrolases/chemistry , Glycoproteins/analysis , Hydrogen Deuterium Exchange-Mass Spectrometry/methods , Animals , Glycosylation , Humans , Mice , Models, Molecular , Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase/chemistry , Peptides/analysisABSTRACT
Bifidobacterium animalis subsp. lactis BB-12 is a widely used probiotic strain associated with a variety of health-promoting traits. There is, however, only limited knowledge available regarding the membrane proteome and the proteins involved in oligosaccharide transport in BB-12. We applied two enrichment strategies to improve the identification of membrane proteins from BB-12 cultures grown on glucose and on xylo-oligosaccharides, the latter being an emerging prebiotic substrate recently reported to be fermented by BB-12. Our approach encompassed consecutive steps of detergent- and carbonate-treatment in order to generate inside-out membrane vesicles and to interfere with binding of membrane-associated proteins to the membrane, respectively. Proteins in the enriched membrane fraction and membrane-associated fraction were digested by lysyl endopeptidase and trypsin followed by peptide sequencing by LC-ESI-Q-TOF MS/MS. Ninety of a total of 248 identified unique proteins were predicted to possess transmembrane segments (TMSs), and 56 of these have more than one TMS. Seventy-nine of the identified proteins are annotated to be involved in transport of amino acids, oligosaccharides, inorganic ions, nucleotides, phosphate or exopolysaccharides, or to belong to the F1F0-ATP-synthetase complex and the protein translocation machinery, respectively.
Subject(s)
Bacterial Proteins/metabolism , Bifidobacterium/metabolism , Proteomics/methods , Computational Biology/methods , Detergents/pharmacology , Glucose/chemistry , Glucose/metabolism , Humans , Oligosaccharides/chemistry , Peptides/chemistry , Probiotics/chemistry , Proteome/metabolism , Serine Endopeptidases/chemistry , Spectrometry, Mass, Electrospray Ionization/methods , Trypsin/chemistryABSTRACT
Beta2-glycoprotein I (beta2GPI) is a five-domain protein associated with the antiphospholipid syndrome (APS), however, its normal biological function is yet to be defined. beta2GPI is N-glycosylated at several asparagine residues and the glycan moiety conjugated to residue 143 has been proposed to interact with the Gly40-Arg43 motif of beta2GPI. The Gly40-Arg43 motif has also been proposed to serve as the epitope for the anti-beta2GPI autoantibody associated with APS. We hypothesized that the structure or composition of the glycan at Asn-143 might be associated with the APS symptom by shielding or exposing the Gly40-Arg43 motif towards the anti-beta2GPI autoantibody. To test this hypothesis we used mass spectrometry (MS) for comparative glycopeptide profiling of human beta2GPI obtained from blood serum from four healthy test subjects and six APS patients. It revealed significant differences in the extent of sialylation and branching of glycans at Asn-143. Biantennary glycans were more abundant than triantennary glycans at Asn-143 in both healthy subjects and patients. In APS patient samples we observed a decrease in sialylated triantennary glycans and an increase in sialylated biantennary glycan structures, as compared to controls. These data indicate that some APS patients have beta2GPI molecules with a reduced number of negatively charged sialic acid units in the glycan structure at Asn-143. This alteration of the electrostatic properties of the glycan moiety may attenuate the intramolecular interactions with the positively charged Gly40-Arg43 motif of beta2GPI and, in turn, leads to conformational instability and exposure of the disease-related linear epitope Gly40-Arg43 to the circulating autoantibody. Thus, our study suggests a link between site-specific glycan profiles of beta2GPI and the pathology of antiphospholipid syndrome.
