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Measurement of mast cell surface molecules by high-throughput immunophenotyping using transcription (HIT)
D James Haddon1, Justin A Jarrell, Michael R Hughes
1Division of Immunology and Rheumatology, Stanford University School of Medicine, CCSR Building, Room 2215A, Mail Code 5166, 269 Campus Drive, Stanford, CA, 94305, USA.
Insights
A new assay, High-Throughput Immunophenotyping using Transcription (HIT), analyzes numerous mast cell proteins simultaneously. This method effectively measures changes in protein levels during cell stimulation.
Area of Science:
- Immunology
- Proteomics
- Cell Biology
Background:
- Mast cells play crucial roles in allergic responses and immunity.
- Characterizing mast cell surface antigens and intracellular molecules is vital for understanding their function.
- Existing proteomic assays may have limitations in throughput and scope.
Purpose of the Study:
- To introduce and validate a highly multiplexed proteomic assay, High-Throughput Immunophenotyping using Transcription (HIT).
- To apply HIT for comprehensive analysis of human mast cell surface antigens and intracellular proteins at rest and upon stimulation.
Main Methods:
- HIT assay involves conjugating barcoded oligonucleotides to Fab fragments of primary antibodies.
- A cocktail of up to 48 barcoded antibodies is used to stain mast cells.
- Barcodes are amplified via in vitro transcription, and transcripts are quantified using microarrays.
Main Results:
- The HIT assay allows simultaneous analysis of up to 100 analytes, including surface antigens and intracellular phosphoproteins.
- Quantified transcript levels correlate well with actual protein levels.
- HIT successfully detected stimulation-dependent changes in mast cell protein expression.
Conclusions:
- HIT is a powerful, broad-spectrum tool for characterizing mast cell surface antigens, signaling molecules, transcription factors, and cytokines.
- This assay enables high-throughput analysis of complex cellular states.
- HIT offers a valuable approach for immunological research and drug discovery.
Abstract:
Here we describe the application of a highly multiplexed proteomic assay, called HIT (high-throughput immunophenotyping using transcription), to analyze human mast cell surface antigens at rest and during stimulation. HIT allows analysis of up to 100 analytes, including surface antigens and intracellular phosphoproteins, transcription factors, and cytokines, in a single experiment. Briefly, anti-mouse monovalent Fab fragments are covalently conjugated with barcoded oligonucleotides to generate a panel of conjugates. The oligonucleotide-Fab fragment conjugates are bound to monoclonal primary antibodies, creating a cocktail of up to 48 unique barcoded primary antibodies. As few as 100,000 mast cells are stained with the cocktail and the barcodes of the bound primary antibodies are amplified by in vitro transcription with fluorescently labeled NTPs. The resulting barcoded transcripts are quantified using a microarray spotted with oligonucleotides that are complementary to the barcoded transcripts. Differences in levels of the barcoded transcripts correlate well with actual protein levels and are capable of detecting stimulation-dependent changes in protein levels. HIT is an invaluable, broad-spectrum approach for characterizing mast cell surface antigens, signaling molecules, transcription factors, and cytokines.
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