Immunoglobulin G (IgG) is a major class of antibodies in the human immune system and plays crucial roles in immune responses against pathogens and other foreign substances. IgG molecules consist of two heavy chains and two light chains, with each heavy chain containing a glycosylation site located at the Fc region. Glycosylation of IgG Fc plays significant roles in modulating immune functions, such as antibody-dependent cellular cytotoxicity (ADCC), complement activation, and inflammatory responses.
The glycan structures attached to the Fc region of IgG molecules are highly heterogeneous and can vary in composition and structure. Alterations in IgG Fc glycosylation have been implicated in various diseases, including autoimmune disorders, infectious diseases, and cancer. For example, changes in IgG Fc glycosylation profiles have been associated with disease progression, treatment response, and prognosis in certain cancers and autoimmune diseases.
Despite the importance of IgG Fc glycosylation in immune regulation and disease pathogenesis, comprehensive analysis of IgG Fc glycopeptides remains challenging due to technical limitations. Traditional methods for glycoprotein analysis often lack the sensitivity, throughput, and reproducibility required for comprehensive characterization of IgG Fc glycosylation patterns.
Liquid chromatography-mass spectrometry (LC-MS) has emerged as a powerful tool for glycoproteomics analysis due to its high sensitivity, resolution, and capability for large-scale analysis. LC-MS-based approaches allow for the identification and quantification of glycopeptides with high precision, enabling comprehensive profiling of IgG Fc glycosylation patterns.
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Case. Metabolic Insights into COVID-19 Infection(1)
Research Background:
The study aims to develop a protocol for the high-throughput analysis of IgG Fc glycopeptides using LC-MS. IgG Fc glycopeptides play crucial roles in immune responses and have implications in various diseases. However, their comprehensive analysis has been challenging due to technical limitations.
Materials and Instruments
In the process of methodological development, this study selected three types of samples for method evaluation, namely human U2OS cells (3*105-9*105), mouse liver (1-3 mg), and OCT-embedded human renal cell carcinoma and adjacent tissues (1-3 mg).
Highlight 1: The first establishment of a seamless system for microscale tissue sample preparation based on PCT. Advantages: Minimal protein loss, allowing for minimal sample analysis, and precise control at each step, minimizing technical errors.
Highlight 2: The first utilization of SWATH technology to analyze human tissue samples and establish protein profiles.
Methods:
The protocol involves several key steps:
- Sample Preparation: Human serum samples containing IgG are processed to extract proteins.
- Glycopeptide Enrichment: IgG Fc glycopeptides are selectively enriched from the protein mixture using specific affinity capture methods.
- LC-MS Analysis: Enriched glycopeptides are analyzed using liquid chromatography-mass spectrometry (LC-MS) to identify and quantify IgG Fc glycopeptides.
- Data Processing and Analysis: Computational tools are used to process LC-MS data, identify glycopeptides, and quantify their abundance.
Experimental Results
Development of the PCT Workflow
Schematic Diagram of the PCT Workflow
The research results demonstrate that the use of the PCT method can significantly reduce the losses incurred during sample transfer in traditional extraction methods. Additionally, this study has optimized an efficient and rapid extraction method, with the entire process taking only 6-8 hours to complete protein sample preprocessing, as follows:
(1) Lysis and extraction: A mixture of 8 M urea and 100 mM ammonium bicarbonate supplemented with a protease inhibitor cocktail, subjected to 50 seconds of high pressure followed by 10 seconds of ambient pressure cycling, for approximately 1 hour.
(2) Simultaneous reduction and alkylation: Conducted at 33°C in a dark environment, taking approximately 5 hours, whereas traditional methods require overnight processing.
Yield and Efficiency of PCT-Assisted Tissue Lysis and Protein Digestion
The PCT method is highly suitable for the extraction of minute samples, yielding peptides with high concentration and strong stability. As depicted in the figure above, the peptide yield from human cell lines is 116 μg/mg, while that from two types of tissue samples is 49 μg/mg each. In contrast, the peptide yield from 100 mg of mouse liver sample using conventional methods is only 30 μg/mg. The technical reproducibility of peptide yield using PCT is characterized by a coefficient of variation (CV) of less than 8%, and the peptides produced exhibit similar length and charge distribution to those obtained by conventional methods, with an increased identification of peptides by 10-20%.
SWATH Mass Spectrometry Acquisition
In this study, the SWATH method was applied to quantify peptides from kidney tissue samples to evaluate the reproducibility of the PCT-SWATH workflow. The results demonstrate that, through fractionation into four aliquots and three technical replicates, 1632 proteins were identified. The CV values for technical replicates ranged between 10-12%, indicating highly stable data performance.
The Reproducibility of PCT-SWATH Mass Spectrometry Acquisition
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Identification of Clinical Disease Molecular Subtype Markers by PCT-SWATH Workflow
In this study, 18 biopsy samples from 9 patients with renal cell carcinoma (RCC), classified pathologically into three subtypes (ccRCC, pRCC, chRCC), were analyzed. A total of 2375 proteins were identified, including 53 proteins reported in the literature as markers for renal cancer.
Quantification of RCC Protein Biomarkers by PCT-SWATH Method
Reference
- Guo, Tiannan, et al. "Rapid mass spectrometric conversion of tissue biopsy samples into permanent quantitative digital proteome maps." Nature medicine 21.4 (2015): 407-413.