Method for determining glycated hemoglobin A (HbA1C) by mass spectrometry

1 . A method for determining glycated hemoglobin A (HbA1c) in a sample, comprising:

(a) providing a sample comprising hemoglobin A (HbA) molecules,

(b) subjecting the sample from step (a) to a partial proteolytic digestion step wherein N-terminal fragments of hemoglobin A β-chain molecules are generated, wherein the partial proteolytic digestion is carried out with the endopeptidase Glu-C in a concentration of about 0.6 mg/mL for a time period of less than about 60 min at a temperature of about 37° C., in a buffer comprising ammonium ions, and stopping the partial proteolytic digestion by adding formic acid in water,

(c) enriching the N-terminal fragments of hemoglobin A β-chain molecules generated in step (b), and

(d) subjecting the sample comprising N-terminal fragments of hemoglobin A β-chain molecules to an analysis by mass-spectrometry (MS) wherein the amount or concentration of HbA1c in the sample is determined,

wherein in the partial proteolytic digestion step (b) about 1% to about 20% of the HbA β-chain molecules present in the sample are digested based on the total amount of HbA β-chain molecules originally present in the sample.

2 . The method of claim 1 , wherein the sample in step (a) is a hemolysed whole-blood sample.

3 . The method of claim 1 , wherein the partial proteolytic digestion in step (b) is carried out with the endopeptidase Glu-C which generates an N-terminal fragment of the hemoglobin A ß-chain molecule having a length in the range of 4-20 amino acids.

4 . The method of claim 1 , wherein the partial proteolytic digestion in step (b) is carried out for a time period of about 15 min to about 45 min.

5 . The method of claim 1 , wherein in enriching step (c), N-terminal fragments of hemoglobin A β-chain molecules are enriched by selective elution from the solid phase.

6 . The method of claim 1 , wherein step (d) comprises an MS/MS analysis.

7 . The method of claim 1 , wherein the ratio of glycated hemoglobin A (HbA1c) to non-glycated hemoglobin A (HbA0) is determined.

8 . The method of claim 1 , wherein the partial proteolytic digestion in step (b) is carried out with the endopeptidase Glu-C, wherein the endopeptidase Glu-C cleaves a hemoglobin A ß-chain molecule after amino acid position 6, 8, or 14 according to SEQ ID NO: 1.

9 . The method of claim 5 , wherein in enriching step (c), N-terminal fragments of hemoglobin A β-chain molecules are enriched by selective elution from the solid phase-in the presence of about 5-20% (v/v) acetonitrile.

10 . The method of claim 1 , wherein the ratio of glycated hemoglobin A (HbA1c) to total hemoglobin A (HbA) is determined.