Bottom-up proteomics, a well-established methodology, is extensively employed for the analysis of protein glycosylation, offering profound insights into the structural and functional aspects of proteins. This approach involves the enzymatic cleavage of glycoproteins into peptides, followed by liquid chromatography (LC) separation and mass spectrometry (MS) detection. The utilization of proteases facilitates the accessibility of glycopeptides for downstream analysis, while LC and MS techniques aid in the separation and characterization of these peptides, respectively.
Protein glycosylation, a post-translational modification, plays a pivotal role in various biological processes, including protein folding, stability, and interaction with other molecules. Among the diverse proteins subject to glycosylation, immunoglobulin G (IgG) stands out due to its critical role in immune response regulation. IgG glycosylation patterns, particularly at the conserved Fc N-glycosylation site, significantly influence the effector functions and pharmacokinetic properties of antibodies.
Distinguishing between glycosylation occurring at the Fab and Fc regions of IgG is imperative, given their distinct functional implications. While Fab glycosylation primarily affects antigen binding, Fc N-glycosylation modulates interactions with various immune receptors, thereby regulating antibody-dependent cellular cytotoxicity (ADCC), complement activation, and antibody half-life. Understanding the differential impact of Fab and Fc glycosylation on IgG functionality is crucial for the development of therapeutic antibodies with optimized efficacy and safety profiles.
Traditional bottom-up proteomics approaches often involve extensive peptide separation using techniques such as reversed-phase LC. However, these methods are time-consuming and unsuitable for analyzing large clinical cohorts comprising thousands of samples. To address this limitation, there is a need for high-throughput strategies specifically tailored for the analysis of IgG glycosylation. Such methods should enable the rapid profiling of glycosylation patterns across diverse IgG isoforms and sample cohorts, facilitating the identification of glycoform variations associated with disease states or therapeutic interventions.
Materials
The materials required for conducting the high-throughput analysis of IgG Fc glycopeptides by LC-MS encompass a range of reagents, consumables, equipment, and computational resources. These components are meticulously selected to ensure the successful execution of the experimental protocol and the accurate interpretation of results.
1. Protein G Sepharose beads (GE Healthcare, Uppsala, Sweden): These beads serve as the affinity matrix for the purification of IgG from serum or plasma samples. Protein G exhibits high affinity for the Fc region of IgG molecules, facilitating their selective capture and subsequent elution for downstream processing.
2. Phosphate-buffered saline (PBS): A buffer solution comprising sodium phosphate, potassium phosphate, and sodium chloride dissolved in water. PBS is used for washing and resuspending Protein G beads during the affinity purification step, ensuring optimal conditions for IgG binding and elution.
3. Formic acid (FA): A strong organic acid essential for the acidic denaturation of proteins and the extraction of peptides from biological samples. A 100 mM FA solution is prepared by diluting concentrated FA with water and is utilized for various steps in the sample preparation process, including protein digestion and LC-MS analysis.
4. Ammonium bicarbonate buffer (ABC): A volatile buffer commonly employed for protein digestion by trypsin. ABC buffer at a concentration of 25 mM and pH 7.8 is used to reconstitute dried samples and maintain suitable conditions for enzymatic cleavage of proteins into peptides.
5. Acetic acid: A weak organic acid used to prepare a 20 mM solution, which may be employed for adjusting the pH of buffers or solvent systems during sample preparation or LC-MS analysis.
6. TPCK-treated trypsin: A modified form of trypsin that has been chemically modified to improve stability and specificity. TPCK (tosylphenylalanylchloromethyl ketone) inhibits chymotrypsin activity, ensuring selective cleavage of peptide bonds at the carboxyl side of lysine and arginine residues. Trypsin is utilized for enzymatic digestion of purified IgG samples into glycopeptides for subsequent analysis by LC-MS.
7. LC-MS system components: The LC-MS setup comprises essential components, including a gradient pump, an isocratic pump, a programmable autosampler, a column oven, and a mass spectrometer. These instruments are integrated to facilitate the separation, ionization, and detection of glycopeptides in biological samples.
8. Computational resources: A computer system equipped with sufficient memory (at least 16GB) and specific software packages is essential for processing and analyzing LC-MS data. Software tools such as LaCyTools, Python (with numpy, scipy, matplotlib, and tkinter libraries), and data conversion utilities are utilized for automated data processing, peak detection, integration, and quality assessment.
9. Consumables: Additional consumables including centrifuge tubes, 96-well filter plates, adhesive tape, polypropylene plates, lint-free paper, and vacuum concentrators are required for sample handling, processing, and storage.
Methods
Affinity Purification of IgG:
- The protocol begins by preparing Protein G Sepharose beads and incubating them with serum or plasma samples containing IgG.
- Following incubation, the supernatant is carefully removed, and the beads are washed with phosphate-buffered saline (PBS) to remove non-specifically bound proteins.
- After washing, the beads are resuspended in PBS, and a portion of this bead suspension is applied to each well of a 96-well filter plate.
- The beads are washed again to remove any remaining contaminants, and then serum or plasma samples are added to each well.
- The plate is sealed and incubated on a shaker to allow IgG binding to the Protein G beads.
- After incubation, the samples are washed to remove unbound proteins, and then eluted from the beads using formic acid.
Sample Preparation for Digestion:
- IgG samples eluted from the Protein G beads are mixed with formic acid and dried to remove excess solvent.
- This dried sample is then reconstituted in a solution containing trypsin for enzymatic digestion.
Trypsin Digestion:
- TPCK-treated trypsin is added to the IgG samples for digestion.
- The samples are incubated to allow trypsin to cleave the peptides from the glycoproteins.
- After digestion, the samples can be stored for further analysis.
Liquid Chromatography-Mass Spectrometry (LC-MS):
- Digested samples are injected into an LC-MS system for glycopeptide separation and mass analysis.
- A gradient pump, isocratic pump, autosampler, and column oven are used for chromatographic separation of glycopeptides.
- The LC system is coupled to a mass spectrometer equipped with an electrospray ionization source and quadrupole-time-of-flight (q-TOF) analyzer for accurate mass determination.
- Samples are nebulized and ionized in the ESI source, and then analyzed in the q-TOF analyzer to obtain mass spectra of glycopeptides.
Data Processing:
- LC-MS data are converted to mzXML format for further processing.
- LaCyTools software is used for automated data analysis, including alignment, calibration, and extraction of glycopeptides.
- Parameters for alignment, calibration, and extraction are adjusted as needed to optimize the analysis.
- The software generates output files containing information on glycopeptide intensity, alignment residuals, quality control metrics, PPM error, and signal-to-noise ratios.
Overall, these methods enable the high-throughput analysis of IgG Fc glycopeptides by LC-MS, providing valuable insights into IgG glycosylation patterns and their potential biological significance.
LC-MS separation of tryptic Fc-glycopeptides of a polyclonal IgG standard.
Reference
- Lauc, Gordan, and Manfred Wuhrer. High-throughput glycomics and glycoproteomics. Springer New York, 2017.
