A liquid chromatography-high-resolution mass spectrometry method for separation and identification of hemoglobin variant subunits with mass shifts less than 1 Da
Background: Identification of hemoglobin (Hb) variants is valuable in clinical testing. A common issue with conventional methods for identifying Hb variants is their subpar ability to provide structural breakdowns of the variants. Reports have surfaced of high-resolution mass spectrometry (HR-MS) me...
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Elsevier
2025-01-01
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| Series: | Journal of Mass Spectrometry and Advances in the Clinical Lab |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2667145X25000021 |
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| author | Ainslie Chen Ryan M. Aquino Hector A. Vidal Carolyn V. Wong Ruben Y. Luo |
| author_facet | Ainslie Chen Ryan M. Aquino Hector A. Vidal Carolyn V. Wong Ruben Y. Luo |
| author_sort | Ainslie Chen |
| collection | DOAJ |
| description | Background: Identification of hemoglobin (Hb) variants is valuable in clinical testing. A common issue with conventional methods for identifying Hb variants is their subpar ability to provide structural breakdowns of the variants. Reports have surfaced of high-resolution mass spectrometry (HR-MS) methods that improve on traditional methods; however, ambiguities may arise without separation of Hb subunits prior to HR-MS analysis. Methods: We report a liquid chromatography-high-resolution mass spectrometry (LC-HR-MS) method to separate several pairs of normal and variant Hb subunits with mass shifts of less than 1 Da and successfully identify them in intact-protein and top-down analyses. LC separation was facilitated by a C4 reversed-phase column. Results: Seven heterozygous Hb variant samples (Hb C with α-thalassemia trait, Hb E, Hb D-Punjab, Hb G-Accra, Hb G-Siriraj, Hb Tarrant, and Hb G-Waimanalo) were selected to demonstrate the LC separation of Hb variant and normal subunits with mass shifts of less than 1 Da. The analytes could be explicitly observed in the deconvoluted MS1 mass spectra. The top-down analysis matched the amino acid sequences of the correct Hb variant subunits. Conclusions: The LC-HR-MS method described can effectively separate and identify Hb subunits, especially when the variant subunits have mass deviations of less than 1 Da from their corresponding normal subunits. With further evaluation to prove the clinical utility, the HR-MS methods including CE-HR-MS have the potential to complement or partially replace conventional methods of Hb variant identification in clinical laboratories. |
| format | Article |
| id | doaj-art-fb18b84d282342fa8f6bc312209f2294 |
| institution | Kabale University |
| issn | 2667-145X |
| language | English |
| publishDate | 2025-01-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Journal of Mass Spectrometry and Advances in the Clinical Lab |
| spelling | doaj-art-fb18b84d282342fa8f6bc312209f22942025-02-06T05:13:00ZengElsevierJournal of Mass Spectrometry and Advances in the Clinical Lab2667-145X2025-01-013517A liquid chromatography-high-resolution mass spectrometry method for separation and identification of hemoglobin variant subunits with mass shifts less than 1 DaAinslie Chen0Ryan M. Aquino1Hector A. Vidal2Carolyn V. Wong3Ruben Y. Luo4Department of Pathology, School of Medicine, Stanford University, Stanford, CA, USAClinical Laboratories, Stanford Health Care, Palo Alto, CA, USAClinical Laboratories, Stanford Health Care, Palo Alto, CA, USAClinical Laboratories, Stanford Health Care, Palo Alto, CA, USADepartment of Pathology, School of Medicine, Stanford University, Stanford, CA, USA; Clinical Laboratories, Stanford Health Care, Palo Alto, CA, USA; Corresponding author at: 3375 Hillview Ave, Palo Alto, CA 94304, USA.Background: Identification of hemoglobin (Hb) variants is valuable in clinical testing. A common issue with conventional methods for identifying Hb variants is their subpar ability to provide structural breakdowns of the variants. Reports have surfaced of high-resolution mass spectrometry (HR-MS) methods that improve on traditional methods; however, ambiguities may arise without separation of Hb subunits prior to HR-MS analysis. Methods: We report a liquid chromatography-high-resolution mass spectrometry (LC-HR-MS) method to separate several pairs of normal and variant Hb subunits with mass shifts of less than 1 Da and successfully identify them in intact-protein and top-down analyses. LC separation was facilitated by a C4 reversed-phase column. Results: Seven heterozygous Hb variant samples (Hb C with α-thalassemia trait, Hb E, Hb D-Punjab, Hb G-Accra, Hb G-Siriraj, Hb Tarrant, and Hb G-Waimanalo) were selected to demonstrate the LC separation of Hb variant and normal subunits with mass shifts of less than 1 Da. The analytes could be explicitly observed in the deconvoluted MS1 mass spectra. The top-down analysis matched the amino acid sequences of the correct Hb variant subunits. Conclusions: The LC-HR-MS method described can effectively separate and identify Hb subunits, especially when the variant subunits have mass deviations of less than 1 Da from their corresponding normal subunits. With further evaluation to prove the clinical utility, the HR-MS methods including CE-HR-MS have the potential to complement or partially replace conventional methods of Hb variant identification in clinical laboratories.http://www.sciencedirect.com/science/article/pii/S2667145X25000021LC-HR-MSHb VariantsIntact-Protein AnalysisTop-down Analysis |
| spellingShingle | Ainslie Chen Ryan M. Aquino Hector A. Vidal Carolyn V. Wong Ruben Y. Luo A liquid chromatography-high-resolution mass spectrometry method for separation and identification of hemoglobin variant subunits with mass shifts less than 1 Da Journal of Mass Spectrometry and Advances in the Clinical Lab LC-HR-MS Hb Variants Intact-Protein Analysis Top-down Analysis |
| title | A liquid chromatography-high-resolution mass spectrometry method for separation and identification of hemoglobin variant subunits with mass shifts less than 1 Da |
| title_full | A liquid chromatography-high-resolution mass spectrometry method for separation and identification of hemoglobin variant subunits with mass shifts less than 1 Da |
| title_fullStr | A liquid chromatography-high-resolution mass spectrometry method for separation and identification of hemoglobin variant subunits with mass shifts less than 1 Da |
| title_full_unstemmed | A liquid chromatography-high-resolution mass spectrometry method for separation and identification of hemoglobin variant subunits with mass shifts less than 1 Da |
| title_short | A liquid chromatography-high-resolution mass spectrometry method for separation and identification of hemoglobin variant subunits with mass shifts less than 1 Da |
| title_sort | liquid chromatography high resolution mass spectrometry method for separation and identification of hemoglobin variant subunits with mass shifts less than 1 da |
| topic | LC-HR-MS Hb Variants Intact-Protein Analysis Top-down Analysis |
| url | http://www.sciencedirect.com/science/article/pii/S2667145X25000021 |
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