Transfusion · 2025
Detecting red blood cell protein antigens by tandem mass spectrometry.
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Chavan Sandip, Majumdar Ramanath, Joshi Neha, Madden Benjamin, Peterson Jane, Tauscher Craig T, Schieber Jennifer E, Norgan Andrew P, Jacob Eapen K, Pandey Akhilesh, Juskewitch Justin E
Аннотация
BACKGROUND: Tandem mass spectrometry (MS/MS) has become a common clinical laboratory testing modality has demonstrated success in distinguishing between small protein variations in transthyretin amyloidosis. Since many common clinically significant RBC antigens are also small protein variations, this study aimed to determine if MS/MS could correctly detect common RBC antigens within the Rh, Kell, Duffy, MNS, Kidd, Diego, and Lutheran blood group systems.
STUDY DESIGN AND METHODS: Residual samples from serotyped/genotyped blood donors at a hospital-based blood donation center from February to August 2021 were analyzed. RBC membrane protein preparations underwent protease digestion prior to MS/MS analysis for RhD, RhCE, Kell, atypical chemokine receptor 1 (Duffy), glycophorin A (MNS), glycophorin B (MNS), urea transporter 1 (Kidd), band 3 anion transport protein (Diego), and basal cell adhesion molecule (Lutheran). Untargeted liquid chromatography (LC)-MS/MS detected protein-specific peptides, while targeted LC-selected reaction monitoring (LC-SRM) detected RBC antigen-containing peptides.
RESULTS: Through the use of multiple proteases, untargeted LC-MS/MS detected protein-specific peptides in all but glycophorin B, with band 3 anion transport protein, basal cell adhesion molecule, Kell, and glycophorin A having the greatest protein sequence coverage. Targeted LC-SRM detected antigen-specific peptides for Cw, Dia/Dib, Kpb, and Wra/Wrb, which agreed with the donors' previous typing results.
DISCUSSION: MS/MS can successfully detect peptides from several blood group systems but only detected a subset of their common RBC antigens. Further sample enrichment and MS/MS detection improvements will need to occur before MS/MS could be considered a clinical RBC phenotyping modality.
STUDY DESIGN AND METHODS: Residual samples from serotyped/genotyped blood donors at a hospital-based blood donation center from February to August 2021 were analyzed. RBC membrane protein preparations underwent protease digestion prior to MS/MS analysis for RhD, RhCE, Kell, atypical chemokine receptor 1 (Duffy), glycophorin A (MNS), glycophorin B (MNS), urea transporter 1 (Kidd), band 3 anion transport protein (Diego), and basal cell adhesion molecule (Lutheran). Untargeted liquid chromatography (LC)-MS/MS detected protein-specific peptides, while targeted LC-selected reaction monitoring (LC-SRM) detected RBC antigen-containing peptides.
RESULTS: Through the use of multiple proteases, untargeted LC-MS/MS detected protein-specific peptides in all but glycophorin B, with band 3 anion transport protein, basal cell adhesion molecule, Kell, and glycophorin A having the greatest protein sequence coverage. Targeted LC-SRM detected antigen-specific peptides for Cw, Dia/Dib, Kpb, and Wra/Wrb, which agreed with the donors' previous typing results.
DISCUSSION: MS/MS can successfully detect peptides from several blood group systems but only detected a subset of their common RBC antigens. Further sample enrichment and MS/MS detection improvements will need to occur before MS/MS could be considered a clinical RBC phenotyping modality.