Mass spectrometry-based methods for the detection of circulating histones H3 and H2B in plasma from sepsis or septic shock (SS) patients
Inventors
GARCÍA GIMÉNEZ, José Luis • ROMÁ MATEO, Carlos • PALLARDÓ CALATAYUD, Federico V.
Assignees
Universitat de Valencia • Centro de Investigacion Biomedica en Red CIBER • Fundacion Incliva
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Publication Number
US-11740245-B2
Publication Date
2023-08-29
Expiration Date
Abstract
The present invention proposes a novel method for the detection of circulating histones H3 and H2B in plasma from sepsis or septic shock (SS) patients, based on mass spectrometry-based methods, in particular based on multiple reaction monitoring targeted mass spectrometry (MRM-MS). Such methods allow quantification of histones using an internal standard and show strong specificity and sensitivity values.
Core Innovation
The disclosed invention provides a mass spectrometry-based method for measuring circulating histone H3 in biological samples from a first human subject suspected of having sepsis or septic shock. The method determines the amount of peptide SEQ ID NO 1 (STELLIR) present in the biological sample after trypsin digestion or treatment using a mass spectrometer, and the circulating histone H3 level or concentration is obtained from an averaged ratio of the peptide SEQ ID NO 1.
The measured histone H3 level is linked to clinical indication by determining whether there is an increase relative to a reference value or relative to the level or concentration of circulating histone H3 from a corresponding biological sample of a second human subject not suffering from sepsis or septic shock. The increase in at least circulating histone H3 is indicative that the first human subject is suffering from sepsis or septic shock.
The disclosed approach also measures circulating histone H2B by determining the amount of peptide SEQ ID NO 2 (LLPGELAK) in the biological sample using a mass spectrometer and obtaining a quantitative level or concentration based on an averaged ratio of SEQ ID NO 2. The overall panel supports distinguishing sepsis and septic shock through biomarker increases in circulating histone H3 and, optionally, circulating histone H2B in plasma, serum, or blood.
Claims Coverage
The document contains one independent claim directed to measuring circulating histone H3 by mass spectrometry using a specified Spike-In peptide and an averaged ratio, with sepsis/septic shock indication based on increase relative to a reference or a non-sepsis/septic-shock control sample. Dependent coverage further adds quantitative reference constraints and expands to circulating histone H2B using a second specified peptide and averaged ratio evaluation.
Mass spectrometry-based measurement of circulating histone H3 for sepsis/septic shock indication
A mass spectrometry-based method for measuring the level or concentration of at least circulating histone H3 in one or more biological samples from a first human subject suspected of having sepsis or septic shock, comprising determining the amount of peptide SEQ ID NO 1 (STELLIR) present in the biological sample after trypsin digestion or treatment using a mass spectrometer, wherein the sample is selected from blood, serum and plasma isolated from the subject, and wherein to measure the level or concentration of circulating histone H3 in the biological sample the averaged ratio of SEQ ID NO 1 is obtained, and wherein an increase in the level or concentration of at least circulating histone H3 relative to a reference value or relative to the level or concentration of circulating histone H3 from a corresponding biological sample of a second human subject not suffering from sepsis or septic shock, is indicative that the first human subject is suffering from sepsis or septic shock.
Quantitative diagnostic constraint for circulating histone H3 reference value and fold increase
The method of the independent claim, wherein a histone H3 reference value of 86.36 ng/mL is used and at least a 1.5-fold increase indicates sepsis or septic shock.
Averaged-ratio quantification of circulating histone H2B using peptide SEQ ID NO 2 for sepsis/septic shock indication
A mass-spectrometry-based method determines circulating histone H2B levels in biological samples from a first human by measuring peptide SEQ ID NO 2 (LLPGELAK) with a mass spectrometer, using the averaged SEQ ID NO 2 ratio to quantify H2B, and identifies sepsis or septic shock when histone H3 and H2B levels increase relative to a reference or to levels in a corresponding sample from a second human.
Quantitative diagnostic constraint for circulating histone H2B reference value and fold increase
The method of the independent claim, wherein a histone H2B reference value of 212.03 ng/mL is used and a measured increase of at least 1.5-fold indicates sepsis or septic shock.
Overall claim coverage focuses on targeted mass spectrometry measurement of circulating histone H3 using peptide SEQ ID NO 1 (STELLIR) with an averaged ratio and sepsis/septic shock indication based on increased levels relative to a reference or to a non-sepsis/septic-shock control sample. Dependent coverage introduces quantitative reference constraints for histone H3 and expands quantification to circulating histone H2B using peptide SEQ ID NO 2 (LLPGELAK) with an averaged ratio, again using increased levels relative to reference or control to indicate sepsis or septic shock.
Stated Advantages
Improved diagnostic performance for distinguishing sepsis/septic shock using ROC analysis based on circulating histone H3 (and optionally H2B) levels.
Prognostic ability to distinguish survivors versus non-survivors within the first 24 hours, with histone H3 performing better than histone H2B in the reported results.
Documented Applications
Diagnostic use to indicate that a first human subject suspected of sepsis or septic shock is suffering from sepsis or septic shock by measuring circulating histone H3 (and optionally circulating histone H2B) from plasma/serum/blood using a targeted MRM-MS assay with specified Spike-In peptides.
Triage/prognosis use within the first 24 hours by distinguishing survivors versus non-survivors using circulating histone H3 (reported as performing better than H2B in the results).
Computer program product use for risk/differential diagnosis based on the biomarker measurements and reported thresholds.
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