Do I Need to Trypsin Digest Before Releasing IgG Glycans With PNGase-F?

Immunoglobulin G (IgG) is the main immunoglobulin in human serum, and its biological activity is modulated by glycosylation on its fragment crystallizable region. Glycosylation of IgGs has shown to be related to aging, disease progression, protein stability, and many other vital processes. A common approach to analyze IgG glycosylation involves the release of the N-glycans by PNGase F, which cleaves the linkage between the asparagine residue and the innermost N-acetylglucosamine (GlcNAc) of all N-glycans except those containing a 3-linked fucose attached to the core GlcNAc. The biological significance of these glycans necessitates the development of accurate methods for their characterization and quantification. Currently, researchers either perform PNGase F deglycosylation on intact or trypsin-digested IgGs. Those who perform PNGase F deglycosylation on trypsin-digested IgGs argue that proteolysis is needed to reduce steric hindrance, whereas the other group states that this step is not needed, and the proteolytic step only adds time. There is minimal experimental evidence supporting either assumption. The importance of obtaining complete glycan release for accurate quantitation led us to investigate the kinetics of this deglycosylation reaction for intact IgGs and IgG glycopeptides. Statistically significant differences in the rate of deglycosylation performed on intact IgGs and trypsin-digested IgGs were determined, and the rate of PNGase F deglycosylation on trypsin-digested IgGs was found to be 3- to 4-times faster than on intact IgG.

Reducing Interferences in Glycosylation Site Mapping.

A current method to locate sites of N-linked glycosylation on a protein involves the identification of deamidated sites after releasing the glycans with peptide-N-glycosidase F (PNGase F). PNGase F deglycosylation converts glycosylated Asn residues into Asp. The 1-Da mass tag created by this process is observable by liquid chromatography-tandem mass spectrometry analysis. A potential interference to this method of N-glycosylation site mapping is the chemical deamidation of Asn residues, which occurs spontaneously and can result in false positives. Deamidation is a pH-dependent process that results in the formation of iso-Asp (i-Asp) and native Asp (n-Asp) by a succinimide intermediate, whereas PNGase F deglycosylation results in the conversion of the glycosylation Asn residue into n-Asp. N-linked glycosylation sites can thus be identified by the presence of a single chromatographic peak corresponding to an n-Asp residue within the consensus sequence Asn-X-Ser/Thr, whereas sites of deamidation led to 2 chromatographic peaks resulting from the presence of n-Asp and i-Asp. The intent of this study is to alert investigators in the field to the potential and unexpected errors resulting from this phenomenon and to suggest a strategy to overcome this pitfall and limit the number of false-positive identifications.

Flash Oxidation (FOX) System: A Novel Laser-Free Fast Photochemical Oxidation Protein Footprinting Platform.

Hydroxyl radical protein footprinting (HRPF) is a powerful and flexible technique for probing changes in protein topography. With the development of the fast photochemical oxidation of proteins (FPOP), it became possible for researchers to perform HRPF in their laboratory on a very short time scale. While FPOP has grown significantly in popularity since its inception, adoption remains limited due to technical and safety issues involved in the operation of a hazardous Class IV UV laser and irreproducibility often caused by improper laser operation and/or differential radical scavenging by various sample components. Here, we present a new integrated FOX (Flash OXidation) Protein Footprinting System. This platform delivers sample via flow injection to a facile and safe-to-use high-pressure flash lamp with a flash duration of 10 µs fwhm. Integrated optics collect the radiant light and focus it into the lumen of a capillary flow cell. An inline radical dosimeter measures the hydroxyl radical dose delivered and allows for real-time compensation for differential radical scavenging. A programmable fraction collector collects and quenches only the sample that received the desired effective hydroxyl radical dose, diverting the carrier liquid and improperly oxidized sample to waste. We demonstrate the utility of the FOX Protein Footprinting System by determining the epitope of TNFα recognized by adalimumab. We successfully identify the surface of the protein that serves as the epitope for adalimumab, identifying four of the five regions previously noted by X-ray crystallography while seeing no changes in peptides not involved in the epitope interface. The FOX Protein Footprinting System allows for FPOP-like experiments with real-time dosimetry in a safe, compact, and integrated benchtop platform.