Determination and characterization of site-specific N-glycosylation using MALDI-Qq-TOF tandem mass spectrometry: case study with a plant protease

MALDI tandem mass spectrometry analysis on a hybrid quadrupole-quadrupole time-of-flight (Qq-TOF) instrument was used in combination with two-dimensional gel electrophoresis, proteolytic digestion, and liquid chromatography for identification and structural characterization of glycosylation in a novel glycoprotein, pathogenesis-related subtilisin-like proteinase P69B from tomato. Glycopeptide fractions from microcolumn reversed-phase HPLC deposited on MALDI targets were identified from MS by their specific m/z spacing patterns (203, 162, 146 u) between glycoforms. In most cases, MS/MS spectra of [M + H]+ ions of glycopeptides featured peaks useful for determining sugar compositions, peptide sequences, and thus probable glycosylation sites. Furthermore, peptide-related product ions could readily be used in database search procedures to identify the glycoprotein. Four out of five predicted glycosylation sites were biologically relevant and occupied by five N-linked glycan side chains each. In addition, the fragmentation efficiency allowed detection of further modification of methionine-containing glycoforms with either oxidized or iodoacetamide alkylated methionine. The high resolution furnished by MALDI-Qq-TOF allowed rapid and sensitive structural characterization of site-specific N-glycosylation from a limited quantity of material and revealed heterogeneity at different levels, including different glycan side-chain modifications, and heterogeneity of oligosaccharide structures on the same glycosylation site.     

Isolation and identification of sialylated glycopeptides from bovine alpha1-acid glycoprotein by off-line capillary electrophoresis MALDI-TOF mass spectrometry

Sialylated glycopeptides contained in liquid chromatographic fractions of bovine alpha1-glycoprotein tryptic digests were isolated from asialo peptides using capillary electrophoresis (CE). CE effluents were deposited directly onto a metallic target and analyzed using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. This method allowed the characterization of four N-glycosylation sites in the glycoprotein, and each site was observed as a set of sialylated peptide glycoforms. Tandem mass spectrometry was used to confirm peptide sequences and glycan content in glycoforms. The CE method developed for this study resulted in a very clear separation of the sialylated from the asialo content of glycoprotein digests and proved very useful in the determination of the nature and location of sialylated glycans along the protein chain. This article is the first report describing the use of on-target CE fraction collection using a MALDI removable sample concentrator.     

Deamidation of -Asn-Gly- sequences during sample preparation for proteomics: Consequences for MALDI and HPLC-MALDI analysis

We find that peptides containing -Asn-Gly- sequences typically show approximately 70-80% degree of deamidation after standard overnight (approximately 12 h) tryptic digestion at 37 degrees C. This emphasizes the need for more detailed information about the deamidation reaction in -Asn-Gly- sequences, in which two deamidated species are produced, one containing an aspartic acid (-Asp-Gly-) residue and the other containing an isoaspartic acid (-betaAsp-Gly-) residue. For the peptide SLNGEWR (54-60 beta-galactosidase, E. coli), all three components of the reaction mixture were separated by HPLC on C18 300-A sorbent, with trifluoroacetic acid as an ion-pairing modifier. Their intensity ratios suggested the elution order -betaAsp-/-Asn-/-Asp-, which was subsequently confirmed by MALDI MS and MS/MS analysis. The kinetics of the deamidation was studied in detail for the synthetic SLNGEWR parent using RP HPLC with UV detection. The half-life of this peptide was found to be approximately 8 h under digestion conditions. Analysis of a large pool of peptide retention data shows that the -betaAsp-/-Asn-/ -Asp- retention order is normally observed under the above conditions, especially if the original -NG- sequence is surrounded by hydrophobic amino acids. However, changing chromatographic conditions to 100-A pore size sorbents, or using formic acid as a modifier, increases the retention time of -betaAsp- relative to the -Asn-/-Asp- pair, so the order can sometimes be different.     

Polysaccharide microarrays for polysaccharide-platelet-derived-growth-factor interaction studies

Polysaccharide microarrays have great potential for the high-throughput analysis of polysaccharide-protein interactions. Here we demonstrate that a polysaccharide microarray prepared by printing a library of dextran polymers derivatized by methylcarboxylate, benzylamide, and sulfate groups (DMCBSu compounds) on to glass slides permitted the rapid identification of a set of compounds able to interact with the platelet-derived growth factor BB (PDGF-BB) isoform, a growth factor involved in wound healing. Microarray interaction results were compared to the capacity of DMCBSu compounds to potentiate the in vitro PDGF-BB-induced proliferation of human dermal fibroblasts.     

Predicting retention time shifts associated with variation of the gradient slope in peptide RP-HPLC

We have developed a sequence-specific model for predicting slopes (S) in the fundamental equation of linear solvent strength theory for the reversed-phase HPLC separation of tryptic peptides detected in a typical bottom-up-proteomics experiment. These slopes control the variation in the separation selectivity observed when the physical parameters of chromatographic separation, such as gradient slope, flow rate, and column size are altered. For example, with the use of an arbitrarily chosen set of tryptic peptides with a 4-times difference in the gradient slope between two experiments, the R(2)-value of correlation between the observed retention times of identical species decreases to ~0.993-0.996 (compared to a theoretical value of ~1.00). The observed retention time shifts associated with variations of the gradient slope can be predicted a priori using the approach described here. The proposed model is based on our findings for a set of synthetic species (Vu, H.; Spicer, V.; Gotfrid, A.; Krokhin, O. V. J. Chromatogr., A, 2010, 1217, 489-497), which postulate that slopes S can be predicted taking into account simultaneously peptide length, charge, and hydrophobicity. Here we extend this approach using an extensive set of real tryptic peptides. We developed the procedure to accurately measure S-values in nano-RP HPLC MS experiments and introduced sequence-specific corrections for a more accurate prediction of the slopes S. A correlation of ~0.95 R(2)-value between the predicted and experimental S-values was demonstrated. Predicting S-values and calculating the expected retention time shifts when the physical parameters of separation like gradient slope are altered will facilitate a more accurate application of peptide retention prediction protocols, aid in the transfer of scheduled MRM (SRM) procedures between LC systems, and increase the efficiency of interlaboratory data collection and comparison.     

Bose-Einstein condensation and superfluidity of trapped polaritons in graphene and quantum wells embedded in a microcavity

The theory for spontaneous coherence of short-lived quasiparticles in two-dimensional excitonic systems is reviewed, in particular, quantum wells (QWs) and graphene layers (GLs) embedded in microcavities. Experiments with polaritons in an optical microcavity have already shown evidence of Bose-Einstein condensation (BEC) in the lowest quantum state in a harmonic trap. The theory of BEC and superfluidity of the microcavity excitonic polaritons in a harmonic potential trap is presented. Along the way, we determine a general method for defining the superfluid fraction in a two-dimensional trap, within the angular momentum representation. We discuss BEC of magnetoexcitonic polaritons (magnetopolaritons) in a QW and GL embedded in an optical microcavity in high magnetic field. It is shown that Rabi splitting in graphene is tunable by the external magnetic field B, while in a QW the Rabi splitting does not depend on the magnetic field in the strong B limit.     

Zirconia nanoceramic via redispersion of highly agglomerated nanopowder and spark plasma sintering

A 2.7 mol% yttria stabilizing tetragonal zirconia (2.7Y-TZP) nanopowder was synthesized and stored for five years. Humidity and unsatisfactory storage conditions gradually caused heavy agglomeration. Within a few months, 2.7Y-TZP nanopowder became useless for any technological application. A bead-milling deagglomeration technique was applied to recover the heavily agglomerated yttria-stabilized zirconia nanopowder. Low-temperature sintering and spark plasma sintering (SPS) were performed, resulting in fully dense nanostructured ceramics. Compacts formed with heavily agglomerated powder present low sinterability and poor mechanical properties. Bead-milling suspension formed compacts exhibit mechanical properties in the range of the values reported for nanostructured zirconia. This observation confirms the effectiveness of bead-milling in the deagglomeration of highly agglomerated nanopowders. The high value of Vickers hardness of 13.6 GPa demonstrates the success of the processing technique for recovering long-time-stored oxide nanopowders.     

Influence of ion irradiation on internal residual stress in DLC films

The dependence of internal residual stress in thin diamond-like carbon films grown on Si substrate by PECVD technique on most important growth parameters, namely RF-power, DC bias voltage and substrate temperature, is described. Results show that compressive stress reaches the highest value of 2.7 GPa at low RF-power and DC bias. Increase of substrate temperature from 250 to 350 °C leads to nonlinear increase of stress value. Inhomogeneity of residual stress along the film surface disappears when film is deposited at temperatures above 275 °C. Post-growth film irradiation by P⁺ and In⁺ ions cause decrease of compressive stress followed by its inversion to tensile. For all ion energy combinations used residual stress changes linearly with normalized fluence up to 0.2 DPA with slope (8.7 ± 1.3) GPa/DPA.